Antibodies to MAGMAS and uses thereof

ABSTRACT

The present invention relates to isolated and purified Magmas protein, isolated and purified nucleic acids encoding Magmas protein, and antibodies to Magmas protein, which interacts with granulocyte macrophage colony stimulating factor (GM-CSF). More specifically, the invention relates to uses of such anti-Magmas antibodies. The invention further relates to a method for diagnosis, prognosis and treatment of diseases, particularly, cancer, Alzheimer&#39;s disease and mitochondrial diseases using Magmas sequences and antibodies directed against the Magmas protein or fragments thereof.

FIELD OF INVENTION

The present invention relates to isolated and purified Magmas protein,isolated and purified nucleic acids encoding Magmas protein, andantibodies to Magmas protein, which interacts with granulocytemacrophage colony stimulating factor (GM-CSF). More specifically, theinvention relates to uses of such anti-Magmas antibodies. The inventionether relates to a method for diagnosis, prognosis and treatment ofdiseases using Magmas sequences and antibodies directed against theMagmas protein.

BACKGROUND OF THE INVENTION

GM-CSF and interleukin 3 (IL-3) are two of many growth factors thataffect the survival, growth and differentiation of hematopoietic cells.The receptors for these factors consist of an α subunit that isresponsible for the specificity of ligand binding and a shared subunitknown as the common β chain (βc). The significant α subunit homologytogether with shared β subunit composition of these receptor complexesaccounts for the similar activities that IL-3 and GM-CSF have onhematopoietic cells.

The signal transduction pathways of IL-3 and GM-CSF are nearly identical[1]. An early event for both following ligand binding is the tyrosinephosphorylation of βC which is believed to be important in theactivation of several signal transduction pathways. Jak2 which isconstitutively bound to βc in the absence of growth factor is one of thekinases responsible for the tyrosine phosphorylation of βc [2].Phosphorylated tyrosine residues on PC allow interactions with proteinscontaining src homology 2 domains (SH2) such as Shc [3,4], Shp-2 [5],and STAT [6]. Activated Jak2 also phosphorylates bound STAT 5 moleculesresulting in homo- or heterodimerization with other STATs and entry intothe nucleus where they act as transcriptional regulators of a variety ofgenes [7]. She interactions with βc leads to recruitment of grb2 andsos[8], and activation of Ras [9] and MAP kinase. GM-CSF and IL-3activation of PI3K [10, 11] also appears to be mediated through βc viainteractions with lyn [12,13], and possibly SHP2 [14]. RACK1 is anothermolecule which is constitutively bound to βc in the absence of ligand[15]. This protein may regulate the activities of the tyrosine kinasesSrc and Lck and protein kinase C. However survival and at least alimited degree of proliferation has been shown to occur in cells with amutant βc that does not contain any tyrosine residues, demonstratingredundancy in the signaling process [16, 17].

Growth factors are essential for the survival, proliferation anddifferentiation of normal and malignant cells. GM-CSF, along with IL-6,EGF, and IGF-1 are among the growth factors known to be importantregulators of carcinoma cells, e.g., prostate carcinoma cells[45][72][73][74][76][47][63]. For example, the GM-CSF receptor isexpressed in prostate carcinoma cell lines and in primary prostate tumor[47][63]. Some prostate carcinomas even produce GM-CSF, which results inautocrine mediated proliferation and growth factor independence[75][77]. Identifying key regulatory proteins and pathways involved inGM-CSF signaling would be useful for the development of new modalitiesfor the detection and treatment of cancer, e.g. prostate cancer.

Cancer of the prostate is the most commonly diagnosed cancer in men andis the second most common cause of cancer death (Carter, et al., 1990;Armbruster, et al., 1993). If detected at an early stage, prostatecancer is potentially curable. However, a majority of cases arediagnosed at later stages when metastasis of the primary tumor hasalready occurred (Wang, et al., 1982). Early diagnosis is problematicbecause the current tests tend to provide a substantial number of falsepositives and many individuals who test positive in these screens do notdevelop cancer. Present treatment for prostate cancer includes radicalprostatectomy, radiation therapy, or hormonal therapy. No systemictherapy has clearly improved survival in cases of hormone refractorydisease. With surgical intervention, complete eradication of the tumoris not always achieved and the observed re-occurrence of the cancer(12-68%) is dependent upon the initial clinical tumor stage (Zietman, etal., 1993). Thus, alternative methods of diagnosis, and treatmentincluding prognosis, prophylaxis or prevention would desirable.

Mitochondria are cellular organelles with various tasks, includingcellular energy production. Therefore, mitochondrial disorders mostcommonly manifest in tissues highly dependent on biological energy: thebrain, heart, muscle and the main sense organs, in particular the eyeand inner ear. Mitochondrial diseases include phenotypes resemblingseveral rather common conditions, such as myopathy, hearing impairment,epilepsy, diabetes, muscle weakness or paralysis.

Mitochondrial disorders can be caused by mutations in the genes inmitochondrial DNA (mtDNA) or nuclear DNA. While mtDNA encodes only 37genes, the number of nuclear DNA genes that encode proteins essentialfor mitochondrial function is unknown. Identification of these novelnuclear genes would be an important step in diagnostic and prognosticanalysis and eventually treatment of mitochondrial diseases associatedwith defects in the proteins encoded by the nuclear genes.

Alzheimer's Disease (“AD”) is a neurodegenerative illness characterizedby memory loss and other cognitive deficits. McKhann et al., Neurology34: 939 (1984). It is the most common cause of dementia in the UnitedStates. AD can strike persons as young as 40-50 years of age, yet,because the presence of the disease is difficult to determine withoutdangerous brain biopsy, the time of onset is unknown. The prevalence ofAD increases with age, with estimates of the affected populationreaching as high as 40-50% by ages 85-90. Evans et al., JAMA 262: 2551(1989); Katzman, Neurology 43: 13 (1993).

By definition, AD is definitively diagnosed through examination of braintissue, usually at autopsy. Khachaturian, Arch. Neurol. 42: 1097 (1985);McKhann et al., Neurology 34: 939 (1984). Neuropathologically, thisdisease is characterized by the presence of neuritic plaques (NP),neurofibrillary tangles (NFT), and neuronal loss, along with a varietyof other findings. Mann, Mech. Ageing Dev. 31: 213 (1985).

Thus far, diagnosis of AD has been achieved mostly through clinicalcriteria evaluation, brain biopsies and post mortem tissue studies.Research efforts to develop methods for diagnosing Alzheimer's diseasein vivo include (1) genetic testing, (2) immunoassay methods and (3)imaging techniques.

SUMMARY OF THE INVENTION

The present invention is directed to antibodies to a newly identifiedprotein encoded by nuclear DNA, and uses of such antibodies. Theinvention further provides the nucleic acid and amino acid sequence ofMagmas and variations of the nucleic acid and amino acid sequence of theMagmas providing useful diagnostic tools for cancer. The invention isbased upon our discovery, purification and isolation of a novelmitochondrial and ribosome associated protein, Magmas, that has a rolein GM-CSF activity that differs from IL-3 and without wishing to bebound by a theory, it also potentially interacts with other signaltransduction pathways.

The antibodies of the present invention are useful, for example, indetecting variations of Magmas expression levels. Variations of theexpression level of Magmas appear to be associated with a number ofdisease states including, mitochondrial myopathy, Alzheimer's diseaseand cancer, particularly prostate cancer, neuroblastoma, Ewings sarcoma,osteosarcoma and leukemia, particularly acute myeloid leukemia (AML) andacute lymphoid leukemia (ALL). We have also discovered that the Magmasis associated with ribosomal subunits, particularly with ribosomalsubunit S19. Therefore, antibodies to Magmas are also useful in methodsof diagnosing diseases related to ribosomal dysfunction such as, DiamondBlackfan anemia. We have also discovered a method for diagnostic andprognostic analysis of any of the above mentioned diseases using theseMagmas antibodies. We have further discovered a method of treating suchcancers using the antibodies directed against the Magmas protein.Additionally, we have identified assays that can be used to screen fornovel compounds that can interact with Magmas. We have also identified amethod of diagnosing and treating mitochondrial disorders associatedwith Magmas. Additionally, the novel Magmas sequence variations of thepresent invention provide diagnostic tool for AML, ALL as well asmitochondrial myopathy.

In one embodiment, the invention provides an isolated and purifiednucleic acid sequence comprising SEQ ID NO: 11 or fragments thereof thatconsist of preferably at least 20, 30, 50, 100, or 500 contiguousnucleic acids of the SEQ ID NO: 11. Nucleic acids encoding a proteinhaving at least 85%, preferably at least 90%, more preferably at least95%, 98%, or 99% homology to SEQ ID NO: 4 are also provided. Homology asused herein is determined using a NCBI BLAST nucleotide homologycomparison program, or “BLAST” using its default settings as provided athttp://www.ncbi.nlm.nih.gov/BLAST.

In another embodiment, the invention provides an isolated and purifiednucleic acid sequence encoding SEQ ID NO: 4 or fragments thereof thatconsist of preferably at least 6, 8, 10, 20, or 50 contiguous aminoacids of SEQ ID NO: 4.

Preferably, the fragments are selected from the group consisting ofamino acids 1-29, 1-32, 1-33, 30-125, 33-125, 34-125, of SEQ ID NO: 4.These sequences are useful, for example, in preparing antibodies todiagnose cancers which are associated with Magmas mutations resulting indeletions in Magmas protein as described below. Vectors comprising thenucleic acid sequences and fragments thereof are also provided.

The invention further provides isolated and purified nucleic acidsequences encoding amino acid sequences of the SEQ ID NO: 5, SEQ ID NO:6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9 and vectors comprising suchsequences.

In another embodiment, the invention provides an isolated and purifiedprotein comprising SEQ ID NO: 4 or a fragment thereof having at least 6contiguous amino acids of SEQ ID NO: 4 or an amino acid sequence havingat least 85%, more preferably at least 90% and most preferably at least95%, 98% or 99% homology to SEQ ID NO: 4 as defined using the BLAST withthe standard settings.

The invention further provides nucleic acids encoding fusion proteinscomprising SEQ ID NO: 4 or a fragment thereof.

In one embodiment, the invention relates to an antibody or antibodyfragment directed against an immunogenic fragment of a Magmas protein.As used herein, the term “Magmas protein” refers to a protein having theamino acid sequence of the SEQ ID NO: 4.

In another embodiment, the invention relates to a complex comprising anantibody or antibody fragment directed against an immunogenic fragmentof a Magmas protein linked to a cytotoxic molecule.

In another embodiment, the invention relates to a complex comprising anantibody or antibody fragment directed against an immunogenic fragmentof a Magmas protein linked to a detectable label. In one embodiment, thedetectable label is a radionuclide.

In yet another embodiment, the invention entails a method of detectingcancer comprising the steps of providing a biological sample from anindividual suspected of having cancer, analyzing the Magmas proteinexpression in the sample, comparing the expression of Magmas protein inthe sample against a normal control, wherein increased expression of theMagmas protein in the sample indicates that the individual is atincreased risk for cancer. The expression of the Magmas protein can beanalyzed using either Magmas mRNA or Magmas protein. In the preferredembodiment the analysis is an immunohistochemical analysis with anantibody directed against Magmas protein. Preferably the cancer isprostate cancer, neuroblastoma, Ewings sarcoma, osteosarcoma andleukemia. Most preferably, the cancer is prostate cancer.

In another embodiment, the invention entails a method of determining theseverity of the cancer comprising the steps of taking a biologicalsample of an individual suspected of having prostate cancer, analyzingthe Magmas protein expression in the sample, comparing the expression ofthe Magmas protein in the sample to a normal sample and/or comparing theexpression of the Magmas protein in the sample to expression in a panelof samples comprising of tissue samples representing different stages ofprostate cancer. The expression profile of the Magmas protein in thesample indicates the stage of prostate cancer and provides prognosticinformation and guidance that can be used, for example in designingtreatment options.

In one embodiment, the invention provides a method of diagnosingAlzheimer's disease in an individual in vivo wherein an anti-Magmasantibody is coupled with a radionuclide and injected to an individual'sbrain wherein reduced labeling of the pyramidal neurons of thehippocampus indicate that the individual is affected with Alzheimer'sdisease. The individual is considered affected when the reduction inlabeling with the anti-Magmas antibody is at least about 50-60% of theamount in a sample from a control individual, i.e. an individual notaffected with Alzheimer's disease.

In yet another embodiment, the invention relates to a method of treatingan individual affected with cancer comprising the steps of administeringa therapeutic Magmas blocking amount of a compound that interacts withMagmas such as a Magmas antibody in a pharmaceutically acceptablecarrier to an individual having prostate cancer.

In another embodiment, the invention relates to a method of treating anindividual affected with prostate cancer comprising the steps ofadministering a therapeutic cancer treating amount ofMagmas-antibody-cytotoxic molecule complex in a pharmaceuticallyacceptable carrier to an individual having prostate cancer.

In one embodiment, the invention relates to a method of diagnosing amitochondrial disorder comprising the steps of providing a biologicalsample from an individual suspected of having a mitochondrial disorder,analyzing the Magmas protein expression in the sample, comparing theexpression of Magmas protein in the sample against a normal control,wherein decreased expression of the Magmas protein or expression of atruncated protein or expression of a protein with a number of mutationsin the sample indicates that the individual has a mitochondrialdisorder. The expression of the Magmas protein can be analyzed usingeither Magmas mRNA or Magmas protein. In the preferred embodiment theanalysis is an immunohistochemical analysis with an antibody directedagainst Magmas protein.

In another embodiment, the invention relates to a method of determiningthe severity of a mitochondrial associated disorder comprising the stepsof taking a biological sample of an individual suspected of having amitochondrial disease, analyzing the Magmas protein expression in thesample, comparing the level of expression of the Magmas protein orexpression of a truncated protein or expression of a protein with anumber of mutations in the sample to a normal sample and/or comparingthe expression of the Magmas protein in the sample to expression in apanel of samples comprising of tissue samples representing differentdegrees of severity of a mitochondrial disorder. The expression profileof the Magmas protein in the sample indicates the stage of mitochondrialdisorder and provides prognostic information and guidance that can beused, for example in designing treatment options.

The invention further provides a method of diagnosing mitochondrialmyopathy by detecting the sequence variation E72G in the SEQ ID NO: 4corresponding to a nucleic acid substitution A247G of SEQ ID NO: 11.

In one embodiment, the invention provides a method of diagnosing AML orALL in an individual. The method comprises obtaining a biologicalsample, preferably a bone marrow sample, from an individual suspected ofhaving AML or ALL and analyzing the size, sequence and/or presence ofthe Magmas protein in the sample.

In one embodiment, a sequence variation of Magmas located betweennucleic acid A132 and G133 in the SEQ ID NO: 11 resulting in a deletionin the SEQ ID NO: 4 indicates that the individual is affected with AMLor ALL. In another embodiment, a sequence variation of Magmas locatedbetween G141 and G142 of SEQ ID NO: 11 resulting in a deletion in theSEQ ID NO: 4 indicates that the individual is affected with AML or ALL.In yet another embodiment, the sequence variation located between G228and A229 of SEQ ID NO: 11 resulting in a deletion in the SEQ ID NO: 4indicates that the individual is affected with AML or ALL. The term“sequence variation” is meant to include all kinds of mutationsincluding deletions, insertions, invertions and single nucleotidesubstitutions. Preferably the sequence variation results in a truncatedMagmas protein.

In one embodiment, the analysis is performed using two antibodies. Firstantibody is directed to an antigenic fragment, preferably the N-terminalpart, of Magmas protein which is present in both a normal and mutantMagmas protein, and the second antibody is directed to an epitope whichis not present in the mutant Magmas. A tissue sample is labeled with thefirst and the second antibody wherein reduced labeling using the secondantibody indicates that the individual is affected with an AML or ALL.Preferably, the antibody against a C-terminal fragment of SEQ ID NO: 4is the C-terminal fragment beginning at amino acid 29 of SEQ ID NO: 4and the N-terminal antibody is an antibody against an N-terminalfragment of SEQ ID NO: 4 before the amino acid 29 of the SEQ ID NO: 4.

Alternatively, the analysis of the Magmas can be performed using DNA orRNA samples and analyzing the Magmas deletion mutations using, forexample PCR-based methods, wherein the deleted sequences are detected,for example using primers flanking the deletion region and whereinamplification product using such primers results in a shorter than thenormal Magmas fragment if the sample contains a deletion-mutant Magmasallele.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a gel electrophoresis of differential display products fromPGMD1 cells grown in the presence of IL-3 or GM-CSF. PGMD1 cells werecultured in the presence of IL-3 (t=0) or GM-CSF for 6, 12 or 24 hoursand the resulting RT-PCR products were separated using polyacrylamidegel electrophoresis. The star (*) to the right of the band at t=12 hrindicates the DNA fragment which was used to isolate Magmas.Representative of three experiments.

FIG. 2 shows a Northern blot hybridization analysis of Magmas expressionin PGMD1 cells. mRNA prepared from cells cultured in IL-3 or for 6, 12,24, 48 or 96 hours in the presence of GM-CSF was resolved by agarose gelelectrophoresis and transferred to a nitrocellulose membrane. Themembrane was first hybridized with a DNA probe to Magmas and thenstripped and hybridized with a probe to GAPDH which was used as thecontrol for loading. Representative of three experiments.

FIGS. 3A and B show a cDNA alignment, sequence analysis, and proteinstructure of Magmas. FIG. 3A provides comparison of cDNA and proteinsequence of human (nucleic acids beginning from nucleotide number 15 ofSEQ ID NO: 11 are shown) and mouse Magmas (SEQ ID NO: 12). The aminoacid single letter code is displayed below the mouse cDNA sequence. Thefive amino acids that differ between murine and human Magmas are in boldprint (glutamine 32 to arginine; glutamine 44 to arginine; arginine 10to lysine; lysine 122 to methionine; lysine 124 to histidine). Aminoacids encoding the leader sequence are underlined.

FIG. 3B is a schematic representation of the human genomic DNA (GDNA)organization and protein structure. The boundaries of exons areindicated by vertical lines with introns drawn as inverted triangles.Lengths are in kilobase (kb) or base pairs (bp). The correspondingMagmas protein schematic representation indicates the relative size andexon derivation of the 5′ and 3′ untranslated regions and codingregions. The leader sequence (black box), and areas of low compositionalcomplexity (hatched boxes) are indicated.

FIG. 4 shows the molecular weight determination of Magmas by Westernblot Protein extracts from wild type PGMD1 cells (lanes 1,7) and cellsexpressing Magmas-GFP fusion protein (lane 2) or GFP (lane 3) wereseparated by SDS-PAGE, transferred to nitrocellulose membranes andincubated with anti-Magmas antisera. After incubating with peroxidaselabelled anti-rabbit Ig and chemiluminescent reagent the blot wasexposed to film and the autoradiograph developed. The blot (containinglanes 1-3) was stripped and reprobed with preimmune sera and anti-GFPantibody (lanes 4-6) in a similar manner. Representative of twoexperiments.

FIGS. 5A-F show the intracellular localization of Magmas. Fluorescencemicrograph of PGMD1 cells incubated with rabbit anti-Magmas antibody(FIG. 5A) or preimmune antibody (FIG. 5B) followed by FITC conjugatedgoat anti-rabbit antibody. The nuclei are stained red by propidiumiodide. PGMD1 cells transfected with a vector containing Magmas-GFPfusion protein (FIG. 5C) or vector containing GFP (FIG. 5D).Fluorescence micrograph of mitrochondrial preparations from Magmas-GFPtransfected cells (FIG. 5E) or GFP transfected cells (FIG. 5F).Representative of three experiments.

FIGS. 6A and B demonstrate that antibody to Magmas specificallyrecognizes a mitochondrial protein in prostate carcinoma by electronmicroscopy. The arrowheads show the mitochondrial location of Magmas,while the arrows show its site of synthesis on the ribosomes.

FIGS. 7A and B show the effect of Magmas expression on growth factorinduced proliferation of PGMD1 cells. FIG. 7A shows a Western blot ofMagmas expression. Protein lysates from untransfected PGMD1 cells orcells transfected with vectors containing sense or antisense Magmas cDNAwere subjected to polyacrylamide gel electrophoresis and the proteinswere transferred to nitrocellulose membranes. The membrane was blottedwith rabbit polyclonal anti-Magmas antibody followed by peroxidaseconjugated goat anti-rabbit antibody and chemilumenescence reagent. FIG.7B shows ³H thymidine incorporation of wild type PGMD1 or cellstransfected with sense or antisense Magmas cDNA (corresponding to FIG.6A) cultured in various concentrations of IL-3 (left panel) or GM-CSF(right panel). Wild type

sense 4

sense 7

antisense 8

antisense 16

Representative of two experiments.

DETAILED DESCRIPTION OF THE INVENTION

We have now identified a mitochondrial associated protein, Magmas, thathas a role in GM-CSF activity that differs from IL-3. The level of thisprotein appears to be associated with cancer, particularly prostatecancer, neuroblastoma, Ewings sarcoma, osteosarcoma and leukemia. Thelevel of Magmas is also affected in patients with Alzheimer's disease.In addition, mutation in Magmas was identified in a patient with amitochondrial disease. Moreover, we have generated and discoveredantibodies against the novel Magmas protein. We have also discovered amethod for diagnostic and prognostic analysis of cancer using theseMagmas antibodies. Any of the above-mentioned cancers can be diagnosedby looking for elevated levels of Magmas. Preferably, the cancer inprostate cancer. We have further discovered a method of treating suchcancers using the antibodies directed against the Magmas protein.Additionally, we have identified assays that can be used to screen fornovel compounds that can interact with magmas. Furthermore, we havediscovered a method of diagnostic and prognostic analysis ofmitochondrial disorders and Alzheimer's disease.

In one embodiment, the invention relates to an anti-Magmas antibodywhich refers to an antibody or antibody fragment directed against animmunogenic fragment of a Magmas protein.

Terms “Magmas” and “Magmas protein” refer to an amino acid sequence ofSEQ ID NO: 4:

(SEQ ID NO: 4) MAKYLAQIIVMGVQVVGRAFARALRQEFAASRAAADARGRAGHRSAAASNLSGLSLQEAQQILNVSKLSPEEVQKNYEHLFKVNDKSVGGSFYLQSKVVRAKERLDEELKIQAQEDREKWQMPHT.

Homologues of Magmas as described by the SEQ ID NO: 4 that are at least85% homologous with the SEQ ID NO: 4 as determined using the NCBI BLASTsequence analysis suit at http://www.ncbi.nlm.nih.gov/BLAST/ are alsoprovided.

FIG. 3B shows a schematic representation of the human genomic DNA (gDNA)organization and protein structure. The boundaries of exons areindicated by vertical lines with introns drawn as inverted triangles.Lengths are in kilobase (kb) or base pairs (bp). The correspondingMagmas protein schematic representation indicates the relative size andexon derivation of the 5′ and 3′ untranslated regions and codingregions. The leader sequence (black box), and areas of low compositionalcomplexity (hatched boxes) are indicated

The nucleic acid encoding the human Magmas, which allows one skilled inthe art to design, for example, PCR primers, is depicted in the SEQ IDNO: 11 below.

(SEQ ID NO: 11) 1 aattcggcac caggggagtt tgagccccgg agcagagcgg ctgccatggccaagtacctg 61 gcccagatca ttgtgatggg cgtgcaggtg gtgggcaggg cctttgcacgggccttgcgg 121 caggagtttg cagccagccg ggccgcagct gatgcccgag gacgcgctggacaccggtct 181 gcagccgctt ccaacctctc cggcctcagc ctccaggagg cacagcagattctcaacgtg 241 tccaagctga gccctgagga ggtccagaag aactatgaac acttatttaaggtgaatgat 301 aaatccgtgg gtggctcctt ctacctgcag tcaaaggtgg tccgcgcaaaggagcgcctg 361 gatgaggaac tcaaaatcca ggcccaggag gacagagaaa aatggcagatgccccatacg 421 tgactgctcg gctccccccg cccaccccgc cgcctctaat ttatagcttggtaataaatt 481 tcttttctgc aaaaaa.

The term “Magmas antigen epitope” as used herein refers to a moleculewhich is capable of immunoreactivity with the anti-Magmas monoclonalantibodies of this invention. Magmas antigen epitopes may compriseproteins, protein fragments, peptides, carbohydrates, lipids, and othermolecules, but for the purposes of the present invention are mostcommonly proteins, short oligopeptides, oligopeptide mimics (i.e.,organic compounds which mimic the antibody binding properties of theMagmas antigen), or combinations thereof. Suitable oligopeptide mimicsare described, for example, in PCT application US91/04282.

Particularly diagnostically useful epitopes include, for example,antibodies directed against the following peptide fragments regiondeleted in the following sequences (SEQ ID NOs: 5-9) as compared to theSEQ ID NO: 4 and antibodies against the peptides of SEQ ID NOs: 5-9:

(SEQ ID NO: 5) MAKYLAQIIVMGVQVVGRVFARALRQEFAFARALRQEFAELZ; (SEQ ID NO:6) MAKYLAQIIVMGVQVVGRAFARALRQEFAASRFQPLRPQPPGGTADSQRV QAEPZ; (SEQ ID NO:7) MAKYLAQIIVMGVQVVGRAFARALRQEFAASRRHSRFSTCPSZ); (SEQ ID NO: 8)MAKYLAQIIVMGVQVVGRAFARALRQEFAASTPZ (SEQ ID NO: 9)MAKYLAQIIVMGVQVVGRAFARALRQEFAASRAEADARGRAGHRSAAASNLSGLSLQEAQQKNYEHLFKVNDKSVGGSFYLQTKVVRAKERLDEELKIQA QEDRKKGQMPHTZ.

Antibodies can be prepared by means well known in the art. The term“antibodies” is meant to include monoclonal antibodies, polyclonalantibodies and antibodies prepared by recombinant nucleic acidtechniques that are selectively reactive with a desired antigen such asMagmas protein or an antigenic epitope thereof.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as Fab,F(ab′)2, Fv, and others which retain the antigen binding function of theantibody. Monoclonal antibodies of any mammalian species can be used inthis invention. In practice, however, the antibodies will typically beof rat or murine origin because of the availability of rat or murinecell lines for use in making the required hybrid cell lines orhybridomas to produce monoclonal antibodies.

As used herein, the term “humanized antibodies” means that at least aportion of the framework regions of an immunoglobulin are derived fromhuman immunoglobulin sequences.

As used herein, the term “single chain antibodies” refer to antibodiesprepared by determining the binding domains (both heavy and lightchains) of a binding antibody, and supplying a linking moiety whichpermits preservation of the binding function. This forms, in essence, aradically abbreviated antibody, having only that part of the variabledomain necessary for binding to the antigen. Determination andconstruction of single chain antibodies are described in U.S. Pat. No.4,946,778 to Ladner et al.

The term “selectively reactive” refers to those antibodies that reactwith one or more antigenic determinants of the desired antigen, e.g.,Magmas protein, and do not react appreciably with other polypeptides.For example, in a competitive binding assay, less than 5% of theantibody would bind another protein, preferably less than 3%, still morepreferably less than 2% and most preferably less than 1%. Antigenicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics. Antibodies can be used for diagnostic applications orfor research purposes.

For example, antibodies may be raised against amino-terminal(N-terminal) or carboxyl-terminal (C-terminal) peptides of a Magmaspolypeptide. Preferably 10-20 N-terminal or 10-25 C-terminal fragmentsof the Magmas protein are used.

One method of generating such an antibody is by using hybridoma mRNA orsplenic mRNA as a template for PCR amplification of such genes [Huse, etal., Science 246:1276 (1989)]. For example, antibodies can be derivedfrom murine monoclonal hybridomas [Richardson J. H., et al., Proc NatlAcad Sci USA Vol. 92:3137-3141 (1995); Biocca S., et al., Biochem andBiophys Res Comm, 197:422-427 (1993) Mhashilkar, A. M., et al., EMBO J.14:1542-1551 (1995)]. These hybridomas provide a reliable source ofwell-characterized reagents for the construction of antibodies and areparticularly useful when their epitope reactivity and affinity has beenpreviously characterized. Another source for such construction includesthe use of human monoclonal antibody producing cell lines. [Marasco, W.A., et al., Proc Natl Acad Sci USA, 90:7889-7893 (1993); Chen, S. Y., etal., Proc Natl Acad Sci USA 91:5932-5936 (1994)]. Another exampleincludes the use of antibody phage display technology to construct newantibodies against different epitopes on a target molecule. [Burton, D.R., et al., Proc Natl Acad Sci USA 88:10134-10137 (1991); Hoogenboom H.R., et al., Immunol Rev 130:41-68 (1992); Winter G., et al., Annu RevImmunol 12:433-455 (1994); Marks, J. D., et al., J Biol Chem 267:16007-16010 (1992); Nissim, A., et al., EMBO J. 13:692-698 (1994);Vaughan T. J., et al., Nature Bio 14:309-314 (1996); Marks C., et al.,New Eng J Med 335:730-733 (1996)]. For example, very large naïve humansFv libraries have been and can be created to offer a large source orrearranged antibody genes against a fragment of Magmas proteins.

Other sources include transgenic mice that contain a humanimmunoglobulin locus instead of the corresponding mouse locus as well asstable hybridomas that secrete human antigen-specific antibodies.[Lonberg, N., et al., Nature 368:856-859 (1994); Green, L. L., et al.,Nat Genet. 7:13-21 (1994)]. Such transgenic animals provide anothersource of human antibody genes through either conventional hybridomatechnology or in combination with phage display technology. In vitroprocedures to manipulate the affinity and fine specificity of theantigen binding site have been reported including repertoire cloning[Clackson, T., et al., Nature 352:624-628 (1991); Marks, J. D., et al.,J Mol Biol 222:581-597 (1991); Griffiths, A. D., et al., EMBO J.12:725-734 (1993)), in vitro afnity maturation [Marks, J. D., et al.,Biotech 10:779-783 (1992); Gram H., et al., Proc Natl Acad Sci USA89:3576-3580 (1992)], semi-synthetic libraries [Hoogenboom, H. R.,supra; Barbas, C. F., supra; Akamatsu, Y., et al., J Immunol151:4631-4659 (1993)] and guided selection [Jespers, L. S., et al., BioTech 12:899-903 (1994)]. Starting materials for these recombinant DNAbased strategies include RNA from mouse spleens [Clackson, T., supra]and human peripheral blood lymphocytes [Portolano, S., et al., supra;Barbas, C. F., et al., supra; Marks, J. D., et al., supra; Barbas, C.F., et al., Proc Natl Acad Sci USA 88: 7978-7982 (1991)].

Thus, one can readily screen an antibody to insure that it has asufficient binding affinity for the Magmas protein or a fragmentthereof. The binding affinity (K_(d)) should be at least about 10⁻⁷l/mol, more preferably at least about 10⁻⁸ l/mol.

For example, cDNA clone in a vector, which are well known to one skilledin the art, encoding Magmas or a fragment thereof may be expressed in ahost using standard techniques such that 5-20% of the total protein thatcan be recovered from the host is the desired protein. Recoveredproteins can be electrophoresed using PAGE and the appropriate proteinband can be cut out of the gel. The desired protein sample can then beeluted from the gel slice and prepared for immunization. Alternatively,a protein of interest can be purified by using conventional methods suchas, for example, affinity chromatography, and molecular sizing, andionic strength separation chromatographic procedures.

Once the protein immunogen is prepared, mice can be immunized twiceintraperitoneally with approximately 50 micrograms of Magmas protein ora fragment thereof per mouse. Sera from such immunized mice can betested for Magmas antibody activity by immunohistology or immunocytologyon any host system expressing such polypeptide and by ELISA with theexpressed polypeptide. For immunohistology, active antibodies of thepresent invention can be identified using a biotin-conjugated anti-mouseimmunoglobulin followed by avidin-peroxidase and a chromogenicperoxidase substrate. Preparations of such reagents are commerciallyavailable; for example, from Zymad Corp., San Francisco, Calif. Micewhose sera contain detectable active Magmas antibodies according to theinvention can be sacrificed three days later and their spleens removedfor fusion and hybridoma production. Positive supernatants of suchhybridomas can be identified using the assays described above and by,for example, Western blot analysis.

To further improve the likelihood of producing a Magmas specificantibody, the amino acid sequence of the polypeptide encoded by aeukaryotic nucleotide sequence of Magmas protein may be analyzed inorder to identify portions of amino acid sequence which may beassociated with increased immunogenicity. For example, polypeptidesequences may be subjected to computer analysis to identify potentiallyimmunogenic surface epitopes. Such computer analysis can includegenerating plots of antigenic index, hydrophilicity, structural featuressuch as amphophilic helices or amphophilic sheets and the like.

For preparation of monoclonal antibodies directed toward polypeptidesencoded by a Magmas sequence of the invention, any technique thatprovides for the production of antibody molecules by continuous celllines may be used. For example, the hybridoma technique originallydeveloped by Kohler and Milstein (Nature, 256: 495-497, 1973), as wellas the trioma technique, the human B-cell hybridoma technique (Kozbor etal., Immunology Today, 4:72), and the EBV-hybridoma technique to producehuman monoclonal antibodies, and the like, are within the scope of thepresent invention. See, generally Larrick et al., U.S. Pat. No.5,001,065 and references cited therein. Further, single-chain antibody(SCA) methods are also available to produce antibodies againstpolypeptides encoded by a eukaryotic nucleotide sequence of theinvention (Ladner et al. U.S. Pat. Nos. 4,704,694 and 4,976,778).

Another method for preparing anti-Magmas antibodies is by in vitroimmunization techniques, such as using spleen cells, e.g., a culture ofmurine spleen cells, injecting an antigen, and then screening for anantibody produced to said antigen. With this method, as little as 0.1micrograms of Magmas antigen can be used, although about 1microgram/milliliter is preferred. For in vitro immunization, spleencells are harvested, for example, mice spleen cells, and incubated atthe desired amount, for example, 1×1 cells/milliliter, in medium pluswith the desired antigen at a concentration typically around 1microgram/milliliter. Thereafter, one of several adjuvants dependingupon the results of the filter immunoplaque assay are added to the cellculture. These adjuvants include N-acetylmuramyl-L-alanyl-D-isoglutamine[Boss, Methods in Enzymology 121:27-33 (1986)], Salmonella typhimuriummitogen [Technical Bulletin, Ribi ImmunoChem. Res. Inc., Hamilton,Mont.] or T-cell condition which can be produced by conventionaltechniques [See, Borrebaeck, C. A. K., Mol. Immunol. 21:841-845 (1984);Borrebaeck, C. A. K., J. Immunol. 136:3710-3715 (1986)] or obtainedcommercially, for example, from Hannah Biologics, Inc. or RibiImmunoChem. Research Inc. The spleen cells are incubated with theantigen for four days and then harvested.

Single cell suspensions of the in vitro immunized mouse spleen cells arethen incubated, for example on antigen-nitrocellulose membranes inmicrofilter plates, such as those available from Millipore Corp. Theantibodies produced are detected by using a label for the antibodiessuch as horseradish peroxidase-labeled second antibody, such as rabbitanti-mouse IgA, IgG, and IgM. In determining the isotype of the secretedantibodies, biotinylated rabbit anti-mouse heavy chain specificantibodies, such as from Zymed Lab., Inc. can be used followed by ahorseradish peroxidase-avidin reagent, such as that available fromVector Lab.

The insoluble products of the enzymatic reaction are visualized as blueplaques on the membrane. These plaques are counted, for example, byusing 25 times magnification. Nitrocellulose membrane of the microfilterplaques readily absorb a variety of antigens and the filtration unitused for the washing step is preferred because it facilitates the plaqueassay.

One then screens the antibodies by standard techniques to findanti-Magmas antibodies of interest. Cultures containing the anti-Magmasantibodies of interest are grown and induced and the supernatants passedthrough a filter, for example, a 0.45 micromiter filter and then througha column, for example, an antigen affinity column or an anti-tag peptidecolumn. The binding affinity is tested using a mini gel filtrationtechnique. See, for example, Niedel, J., Biol. Chem. 256:9295 (1981).One can also use a second assay such as a radioimmunoassay usingmagnetic beads coupled with, for example, anti-rabbit IgG to separatefree ¹²⁵I-labeled antigen from ¹²⁵I-labeled antigen bound by rabbitanti-tag peptide antibody. In a preferred alternative one can measure“on” rates and “off” rates using, for example, a biosensor-basedanalytical system such as “BIAcore” from Pharmacia Biosensor AB [See,Nature 361:186-187 (1993)].

This latter technique requires less antigen than the in vivoimmunization because the in vivo method typically requires about 50micrograms of antigen per mouse per injection and there are usually twoboosts following primary immunization for the in vivo method.

Using any of these antibodies, one can construct V_(H) and V_(L) genes.For instance, one can create V_(H) and V_(L) libraries from murinespleen cells that have been immunized either by the above-described invitro immunization technique or by conventional in vivo immunization andfrom hybridoma cell lines that have already been produced or arecommercially available. One can also use commercially available V_(H)and V_(L) libraries. One method involves using the spleen cells toobtain mRNA which is used to synthesize cDNA. Double stranded cDNA canbe made by using PCR to amplify the variable region with a degenerativeN terminal V region primer and a J region primer or with V_(H) familyspecific primers, e.g., mouse-12, human-7.

For example, the genes of the V_(H) and V_(L) domains of the desiredantibody, such as one to Magmas, can be cloned and sequenced. The firststrand cDNA can be synthesized from, for example, total RNA by usingoligo dT priming and the Moloney murine leukemia virus reversetranscriptase according to known procedures. This first strand cDNA isthen used to perform PCR reactions. One would use typical PCRconditions, for example, 25 to 30 cycles using e.g. Vent polymerase toamplify the cDNA of the immunoglobulin genes. DNA sequence analysis isthen performed. [Sanger, et al., Proc. Natl. Acad. Sci. USA 79:5463-5467(1977)].

Both heavy chain primer pairs and light chain primer pairs can beproduced by this methodology. One preferably inserts convenientrestriction sites into the primers to make cloning easier.

Thereafter, the variable region is chosen. This is then added to the“humanized” framework motif by standard techniques.

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant anti-Magmasantibodies of the invention. See, for example, “Conjugate Vaccines”,Contributions to Microbiology and Immunology, J. M. Cruse and R. E.Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents ofwhich are incorporated herein by reference.

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the anti-Magmas antibodies of the present invention, to othermolecules. For example, representative coupling agents can includeorganic compounds such as thioesters, carbodiimides, succinimide esters,diisocyanates, glutaraldehydes, diazobenzenes and hexamethylenediamines. This listing is not intended to be exhaustive of the variousclasses of coupling agents known in the art but, rather, is exemplary ofthe more common coupling agents. (See, e.g. Killen and Lindstrom 1984,“Specific killing of lymphocytes that cause experimental AutoimmuneMyasthenia Gravis by toxin-acetylcholine receptor conjugates.” Jour.Immun. 133:1335-2549; Jansen, F. K., H. E. Blythman, D. Carriere, P.Casella, O. Gros, P. Gros, J. C. Laurent, F. Paolucci, B. Pau, P.Poncelet, G. Richer, H. Vidal, and G. A. Voisin. 1982. “Immunotoxins:Hybrid molecules combining high specificity and potent cytotoxicity”.Immunological Reviews 62:185-216; and Ghetie and Vitetta, Chemicalconstruction of immunotoxins. Mol. Biotechnol. 2001 July;18(3):251-68.).

Preferred linkers are described in the literature. See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, Umemotoet al. U.S. Pat. No. 5,030,719, describing use of halogenated acetylhydrazide derivative coupled to an antibody by way of an oligopeptidelinker. Particularly preferred linkers include: (i) EDC(1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii)SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS(N-hydroxysulfosuccinimide: Pierce Chem. Co.,Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

In another embodiment, the invention relates to a complex comprising anantibody or antibody fragment directed against an immunogenic fragmentof a Magmas protein linked to a cytotoxic molecule. Variousimmunoconjugates in which antibodies were used to targetchemotherapeutic drugs (P. N. Kularni, A. H. Blair, T. I. Ghose, CancerRes. 41, 2700 (1981); R. Arnon, R. and M. Sela, Immunol. Rev. 62, 5(1982); H. M. Yang and R. A. Resifeld, Proc. Natl. Acad. Sci. U.S.A.,85, 1189 (1988); R. O. Dilman, D. E. Johnson, D. L. Shawler, J. A.Koziol, Cancer Res. 48, 6097 (1988); L. B. Shih, R. M. Sharkey, F. J.Primus, D. M. Goldenberg, Int. J. Cancer 41, 832 (1988); P. A. Trail, etal., Cancer Res. 52, 5693 (1992)), or plant and bacterial toxins (I.Pastan, M. C. Willingham, D. J. Fitzgerald, Cell 47, 641 (1986); D. D.Blakey, E. J. Wawrzynczak, P. M. Wallace, P. E. Thorpe, in MonoclonalAntibody Therapy Prog. Allergy, H. Waldmann, Ed. (Karger, Basel, 1988),pp. 50-90) have been evaluated in preclinical models and found to beactive in vitro and in vivo. A U.S. Pat. No. 5,869,045 describes indetail how to make such antibody conjugates and is hereby incorporatedas reference in its entirety.

Examples of therapeutic agents that can be conjugated with anti-Magmasantibodies include, but are not limited to, antimetabolites, alkylatingagents, anthracyclines, and antimitotic agents. Antimetabolites includemethotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine. Alkylating agents include mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin. Anthracyclinesinclude daunorubicin (formerly daunomycin) and doxorubicin (alsoreferred to herein as adriamycin). Additional examples includemitozantrone and bisantrene. Antimitotic agents include vincristine andvinblastine (which are commonly referred to as vinca alkaloids). Othercytotoxic agents include procarbazine, hydroxyurea, asparaginase,corticosteroids, mytotane (O,P′-(DDD)), and interferons. Furtherexamples of cytotoxic agents include, but are not limited to, ricin,doxorubicin, taxol, cytochalasin B, gramicidin D, ethidium bromide,etoposide, tenoposide, colchicin, dihydroxy anthracin dione,1-dehydrotestosterone, and glucocorticoid. Clearly analogs and homologsof such therapeutic and cytotoxic agents are encompassed by the presentinvention. For example, the chemotherapuetic agent aminopterin has acorrelative improved analog namely methotrexate. Further, the improvedanalog of doxorubicin is an Fe-chelate. Also, the improved analog for1-methylnitrosourea is lomustine. Further, the improved analog ofvinblastine is vincristine. Also, the improved analog of mechlorethamineis cyclophosphamide.

Anti-Magmas antibodies of the present invention can be detected byappropriate assays, e.g., conventional types of immunoassays. Forexample, a sandwich assay can be performed in which Magmas protein or afragment thereof is affixed to a solid phase. Incubation is maintainedfor a sufficient period of time to allow the antibody in the sample tobind to the immobilized polypeptide on the solid phase. After this firstincubation, the solid phase is separated from the sample. The solidphase is washed to remove unbound materials and interfering substancessuch as non-specific proteins which may also be present in the sample.The solid phase containing the anti-Magmas antibody of interest bound tothe immobilized Magmas polypeptide is subsequently incubated withlabeled antibody or antibody bound to a coupling agent such as biotin oravidin. Labels for antibodies are well-known in the art and includeradionuclides, enzymes (e.g. maleate dehydrogenase, horseradishperoxidase, glucose oxidase, catalase), fluorescent molecules(fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine),biotin, and the like. The labeled antibodies are incubated with thesolid and the label bound to the solid phase is measured, the amount ofthe label detected serving as a measure of the amount of anti-ureatransporter antibody present in the sample. These and other immunoassayscan be easily performed by those of ordinary skill in the art.

The resultant antibody can be expressed in vivo or in vitro by a vectorcontaining a DNA segment encoding the single chain antibody describedabove. These can include vectors, liposomes, naked DNA,adjuvant-assisted DNA, gene gun, catheters, etc. Vectors includechemical conjugates such as described in WO 93/04701, which have atargeting moiety (e.g. a ligand to a cellular surface receptor), and anucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNAor RNA viral vector), fusion proteins such as described in PCT/US95/02140 (WO 95/22618) which is a fusion protein containing a targetmoiety (e.g. an antibody specific for a target cell) and a nucleic acidbinding moiety (e.g. a protamine), plasmids, phage, etc. The vectors canbe chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector [Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A.:90 7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci. USA: 87: 1149 (1990)], Adenovirus Vectors[LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet. 3: 219 (1993); Yang, et al., J. Virol. 69: 2004 (1995)] andAdeno-associated Virus Vectors [Kaplitt, M. G., et al., Nat. Genet.8:148 (1994)].

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofMagmas protein in a sample. The antibody can also be used to try to bindto and disrupt Magmas interaction.

The vector can be employed to target essentially any desired targetcell, such as a prostate cancer cell, neuroblastoma cell, Ewings sarcomacell, osteosarcoma cell or leukemia cell. For example, stereotaxicinjection can be used to direct the vectors (e.g. adenovirus, HSV) to adesired location. Additionally, the particles can be delivered byintracerebroventricular (icv) infusion using a minipump infusion system,such as a SynchroMed Infusion System. A method based on bulk flow,termed convection, has also proven effective at delivering largemolecules to extended areas of the brain and may be useful in deliveringthe vector to the target cell Hobo et al., Proc. Natl. Acad Sci USA91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266: 292-305(1994)). Other methods that can be used include catheters, intravenous,parenteral, intramuscular, intraperitoneal and subcutaneous injection,and oral or other known routes of administration.

The antibody cassette is delivered to the cell by any of the knownmeans. For example, a cassette containing these antibody genes, such asthe sFv gene, can be targeted to a particular cell by the techniquesdescribed above.

The vectors may use, for example, internal ribosome entry site (IRES)sequences to force expression of the desired gene, for example, an sFv.An IRES can be used to force a stoichiometric expression of light chainand heavy chain. This forced expression avoids the problem of“silencing” where cells expressing the desired protein arephenotypically not seen, which may occur with a wide range of geneproducts. The IRES sequences can be used to target the single chainantibodies of interest and can be linked with a selectable marker.Selectable markers are well known in the art, e.g., genes that expressprotein that change the sensitivity of a cell to stimuli such as anutrient, an antibiotic, etc. Examples of these genes include neopuro,tk, multiple drug resistance (MDR), etc.

The resultant products of that IRES linkage are not fusion proteins, andthey exhibit their normal biological function. Accordingly, the use ofthese vectors permits the forced expression of a desired protein.Intracellular immunization strategies that are aimed at inhibitingMagmas gene expression can be RNA (antisense, ribozymes, RNA decoys) orprotein (antibodies expressed intracellularly, dominant-negativemutants) based and each group of inhibitors has advantages andlimitations. While RNA based strategies are often limited by theinability to achieve high levels of inhibitor expression or to allowaccurate subcellular localization, protein based strategies may belimited by their potential immunogenicity. Like its normal cellularprotein counterparts, the intracellularly expressed protein transgenewill be degraded by the proteasome and presented on the cell surface byMHC-I to antigen presenting cells [Goldberg, A. L. Functions of theproteasome: The Lysis at the end of the tunnel. Science 268, 522-523(1995); Rock, K. L. A new foreign policy: MRC class I molecules monitorthe outside world. Immunology Today 17, 131-137 (1996)]. When the MHC-Ipresented peptides are recognized as foreign, a subsequent cellularimmune response can be elicited against the transduced cells. Indeed,while results of several cancer gene therapy marking studies [Brenner,M. K., et al. Gene-marking to trace origin of relapse after autologousbone-marrow transplantation. The Lancet 341, 85-86 (1993a); Brenner, M.K., et al. Gene marking to determine whether autologous marrow infusionrestores long-term haemopoiesis in cancer patients. The Lancet 342,1134-1137 (1993b)] and gene replacement studies [Bordignon, C., et al.Gene therapy in peripheral blood lymphocytes and bone marrow for ADAimmunodeficient patients. Science 270, 470-475 (1995); Blaese, R. M., etal.

Using any suitable technique known in the art, such as Northernblotting, quantitative PCR methods, etc. the level of the Magmas proteinor mRNA in cells, particularly in potentially malignant cells such asprostate cells, can be measured. An increase in the level of expressionof Magmas is associated with malignancy or susceptibility formalignancy.

Alternatively, the antibodies of the invention can be used in standardtechniques such as Western blotting or immunohistochemistry to detectthe presence of cells expressing Magmas, to quantify the level ofexpression.

Depending on the particular embodiment of the invention, one or more ofthe antibodies will be coupled with a detectable label such as anenzyme, radioactive isotope, fluorescent compound, chemiluminescentcompound, or bioluminescent compound. Those of ordinary skill in the artwill know of other suitable labels for binding to the antibodies or willbe able to ascertain such using routine experimentation. Furthermore,the binding of these labels to the antibodies can be done using standardtechniques commonly known to those of ordinary skill in the art.

For example, the antibodies can be bound to an enzyme. This enzyme, inturn, when later exposed to its substrate will react to the substrate insuch a manner as to produce a chemical moiety which can be detected, as,for example, spectrophotometric or fluorometric means. Examples ofenzymes that can be used to detectably label are malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, alkaline phosphatase, asparaginase, glucose oxidase,beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, and acetylcholine esterase.

The presence of an antibody can also be detected by labeling theantibody with a radioactive isotope. The presence of the radioactiveisotope could then be determined by such means as the use of a gammacounter or a scintillation counter. Isotopes which are particularlyuseful are ³H, ¹²⁵I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶CI, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se,^(99m)Tc and ¹⁵²Eu.

It is also possible to detect the presence of the antibody by labelingit with a fluorescent compound. When the fluorescently labeled antibodyis exposed to light of the proper wavelength, its presence can then bedetected due to fluorescence of the dye. Among the most importantfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde,and fluorescamine.

Another way in which the antibody can be detectably labeled is bycoupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged antibody is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds are luminol, isoluminol, aromatic-acridinium ester, imidazole,acridinium salt, and oxalate ester.

Likewise, a bioluminescent compound may also be used to label theantibody. Bioluminescence is a special type of chemiluminescence whichis found in biological systems and in which a catalytic proteinincreases the efficiency of the chemiluminescent reaction. The presenceof a bioluminescent binding partner would be determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase, and aequorin.

The antibodies for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of saidcontainer means comprising one of the separate elements to be used inthe method.

For example, one of the container means may comprise a first antibodybound to an insoluble or partly soluble carrier. A second container maycomprise soluble, detectably-labeled second antibody, in lyophilizedform or in solution. The carrier means may also contain a thirdcontainer means comprising a detectably-labeled third antibody inlyophilized form or in solution. Such a kit can be used for sandwichassays. See, e.g., David et al. U.S. Pat. No. 4,376,110 hereinincorporated by reference.

In addition, the carrier means may also contain a plurality ofcontainers each of which comprises different, predetermined amounts ofknown Magmas antigen. These latter containers can then be used toprepare a standard curve into which can be interpolated the resultsobtained from the sample containing the unknown amount of Magmasantigen.

In one embodiment, the invention provides a method of diagnosingAlzheimer's disease (AD) in vivo comprising administering an antibody toMagmas in a suitable carrier to an individual suspected of beingaffected with AD and detecting the amount of Magmas in the pyramidalneurons of the hippocampus wherein decrease in the amount of Magmascompared to a normal brain indicates that the individual is affectedwith an AD.

In an alternative method, the antibodies of the present invention can beused to detect AD in a postmortem tissue sample comprising the pyramidalneurons of the hippocampus.

In vitro imaging can be done with the labels mentioned previously. Invivo imaging is done with diagnostically effective labeled antibodies.The term “diagnostically effective” means that the amount of detectablylabeled antibody administered is sufficient to enable detection of thesite of pyramidal neurons of the hippocampus when compared to abackground signal. Reduction of at least about 40%, preferably about50-60% in labeling using Magmas antibodies as compared to a control isconsidered as indicative of Alzheimer's disease.

Generally, the dosage of detectably labeled antibody for diagnosis willvary depending on considerations such as age, condition, sex, and extentof disease in the patient, contraindications, if any, and othervariables, to be adjusted by the individual physician. Dosage can varyfrom 0.01 mg/kg to 2,000 mg/kg, preferably 0.1 mg/kg to 1,000 mg/kg.

The term “diagnostically labeled” means that the immunoglobulin hasattached to it a diagnostically detectable label. There are manydifferent imaging labels and methods of labeling known to those ofordinary skill in the art. Examples of the types of labels which can beused in the present invention include radioactive isotopes andparamagnetic isotopes.

For diagnostic in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given radionuclide. Theradionuclide chosen must have a type of decay which is detectable for agiven type of instrument. In general, any conventional method forvisualizing diagnostic imaging can be utilized in accordance with thisinvention.

Another important factor in selecting a radionuclide for in vivodiagnosis is that the half-life of a radionuclide be long enough so thatit is still detectable at the time of maximum uptake by the target, butshort enough so that deleterious radiation upon the host is minimized.Ideally, a radionuclide used for in vivo imaging will lack a particulateemission, but produce a large number of photons in a 140-200 keV range,which may be readily detected by conventional gamma cameras.

For in vivo diagnosis, radionuclides may be bound to antibody eitherdirectly or indirectly by using an intermediary functional group.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraceticacid (EDTA). Typical examples of metallic ions which can be bound toimmunoglobulins are ^(99m)Tc, ¹²³I, ¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, ¹²⁵I,⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

The antibodies used in the method of the invention can also be labeledwith paramagnetic isotopes for purposes of in vivo diagnosis. Elementswhich are particularly useful (as in Magnetic Resonance Imaging (Mtechniques) in this manner include ¹⁵⁷Gd, ⁵⁵MN, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe.

Preparations of the imaging antibodies for parenteral administrationinclude sterile aqueous or nonaqueous solutions, suspensions, andemulsions. Examples of non-aqueous solvents are propyleneglycol,polyethyleneglycol, vegetable oil such as olive oil, and injectableorganic esters such as ethyloleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media, parenteral vehicles including sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers, such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like (see, Remington: The Science andPractice of Pharmacy, by Alfonso R. Gennaro, ed. A. L. Gennaro,Lippincott, Williams & Wilkins; ISBN: 0683306472; 20th edition, Dec. 15,2000)

The invention also provides a method of detecting susceptibility forcancer. Preferably the cancer is prostate cancer, neuroblastoma, Ewing'ssarcoma, osteocarcinoma or leukemia. Most preferably the cancer isprostate cancer, AML or ALL. The tissue sample may be a blood sample ora biopsy of the tumor.

In one embodiment, the invention provides a method of diagnosing AMLand/or ALL by using two different antibodies to Magmas. We identifiedseveral Magmas mutations in the primary diagnostic bone marrow samplesfrom patients affected with AML and ALL. All the mutations resulted inC-terminally truncated Magmas protein. Therefore, one embodiment of theinvention is directed to a method comprising providing a tissue sample,for example a bone marrow sample from an individual suspected of beingaffected with leukemia, providing a first antibody binding to anantigenic epitope of the N-terminal part of the Magmas labeled with afirst label, for example a first fluorescent molecule, and providing asecond antibody directed against an antigenic epitope of the C-terminalpart of the Magmas protein labeled with a second label, for example, asecond fluorescent label. Reduction in the C-terminal antibody labelingcompared to a control is indicative of diagnosis of AML and/or ALL.Preferably, the diagnosis is made if the N-terminal antibody is detectedin the tissue sample from an individual suspected of being affected withAML or ALL but the amount of the C-terminal antibody labeling is reducedpreferably at least about 50% compared to a control sample comprising ofnormal bone marrow.

Because early termination often results in unstable transcripts, oneembodiment of the invention is also directed to measuring Magmastranscript levels in a tissue sample from an individual suspected ofbeing affected with AML or ALL. Reduction of the amount of Magmastranscript is indicative of the individual being affected with AML orALL.

The expression of Magmas in the tissue sample can be analyzed by anymeans known to one skilled in the art. These methods include qualitativeor quantitative analysis of Magmas mRNA levels or protein levels. ThemRNA levels can be measured using in situ hybridization techniques asRT-PCR based quantitation methods. Antibodies can be used in a varietyof immunohistochemical methods. Exemplary protocols for all the abovelisted methods can be found in Sambrook and Russel, MOLECULAR CLONING: ALABORATORY MANUAL, 3rd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001).

To compare the expression of Magmas in a cancer sample for diagnostic orprognostic purposes, a number of control samples may be used. One canuse a sample from a healthy individual or alternatively a sample from anon-asserted tissue of the individual suspected of having cancer. Onecan also use a panel of samples from tumors or a reference standardbased upon such panel that have been determined to represent differentdiagnostic stages of the cancer. One example of these is the Gleasonscore classification of prostate cancer Gleason DF. The Veteran'sAdministration Cooperative Urologic Research Group: histologic gradingand clinical staging of prostatic carcinoma. In Tannenbaum M (ed.)Urologic Pathology: The Prostate. Lea and Febiger, Philadelphia, 1977;171-198.

The individual has susceptibility to cancer, particularly prostatecancer, if the expression of Magmas is increased at least by about 5%,more preferably by at least about 10-50%, most preferably over 100%.

In yet another embodiment, the invention relates to a method of treatingan individual affected with prostate cancer comprising the steps ofadministering a therapeutic Magmas blocking amount of a compound thatinteracts with Magmas such as, an antibody to Magmas or an antigenicepitope thereof in a pharmaceutically acceptable carrier to anindividual having prostate cancer.

The invention also relates to a method of treating an individualaffected with a Magmas associated cancer, such as prostate cancer,comprising the steps of administering a therapeutic cancer treatingamount of Magmas-antibody-cytotoxic molecule complex in apharmaceutically acceptable carrier to an individual having prostatecancer.

The antibody may be administered, for example, intradermally,intramuscularly, subcontaneously, or mucosally. The anti-Magmasantibodies when used for administration are prepared under asepticconditions with a pharmaceutically acceptable carrier or diluent.

Doses of the pharmaceutical compositions will vary depending upon thesubject and upon the particular route of administration used. Dosagescan range from 0.1 to 100,000 μg/kg a day, more preferably 1 to 10,000μg/kg.

Routes of administration include oral, parenteral, rectal, intravaginal,topical, nasal, direct injection, etc.

An exemplary pharmaceutical composition is a therapeutically effectiveamount of an oligomer, antibody etc., that recognizes the Magmasprotein, or that can induce an immune reaction against Magmas expressingcells, thereby acting as a prophylactic immunogen, optionally includedin a pharmaceutically-acceptable and compatible carrier. The term“pharmaceutically-acceptable and compatible carrier” as used herein,includes one or more compatible solid or liquid filler diluents orencapsulating substances that are suitable for administration to a humanor other animal. In the present invention, the term “carrier” thusdenotes an organic or inorganic ingredient, natural or synthetic, withwhich the molecules of the invention are combined to facilitateapplication. The term “therapeutically-effective amount” is that amountof the present pharmaceutical composition which produces a desiredresult or exerts a desired influence on the particular condition beingtreated. For example, the amount necessary to raise an immune reactionto provide prophylactic protection. Typically when the composition isbeing used as a prophylactic immunogen at least one “boost” will beadministered at a periodic interval after the initial administration.Various concentrations may be used in preparing compositionsincorporating the same ingredient to provide for variations in the ageof the patient to be treated, the severity of the condition, theduration of the treatment and the mode of administration.

One can prepare kits containing the anti-Magmas antibody or antigen. Thekits would contain, for example, the anti-Magmas antibody in sterile andpyrogen free containers. Doses of the pharmaceutical compositions of theinvention (e.g. the anti-Magmas antibody) will vary depending on thesubject and upon the particular route of administration used. Dosagescan range from 0.1 to 100,000 μg/kg per day, more preferably 1 to 10,000μg/kg. By way of an example only, an overall dose range of from about,for example, 1 microgram to about 300 micrograms might be used for humanuse. This dose can be delivered at periodic intervals based upon thecomposition. For example on at least two separate occasions, preferablyspaced apart by about 4 weeks. In the embodiment where the prime is theanti-Magmas antibody directed to the 10-25 N-terminal amino acids of theMagmas protein, with the boost of anti-Magmas antibody directed to the10-25 C-terminal amino acids of the Magmas protein, it is presentlypreferred to have a series of at least 2 boosts, preferably 3 to 5boosts spread out over a year. Other compounds might be administereddaily. Pharmaceutical compositions of the present invention can also beadministered to a subject according to a variety of other,well-characterized protocols. For example, certain currently acceptedimmunization regimens can include the following: (i) administrationtimes are a first dose at elected date; a second dose at 1 month afterfirst dose; and a third dose at a subsequent date, e.g., 5 months aftersecond dose. See Product Information, Physician's Desk Reference, MerckSharp & Dohme (1990), at 144243. (e.g., Hepatitis B Vaccine-typeprotocol); (ii) for example with other vaccines the recommendedadministration for children is first dose at elected date (at age 6weeks old or older); a second dose at 4-8 weeks after first dose; athird dose at 48 weeks after second dose; a fourth dose at 6-12 monthsafter third dose; a fifth dose at age 4-6 years old; and additionalboosters every 10 years after last dose. See Product Information,Physician's Desk Reference, Merck Sharp & Dohme (1990), at 879 (e.g.,Diphtheria, Tetanus and Pertussis-type vaccine protocols). Desired timeintervals for delivery of multiple doses of a particular composition canbe determined by one of ordinary skill in the art employing no more thanroutine experimentation.

In yet another embodiment, the invention relates to a method ofdiagnosing a mitochondrial disorder in an individual suspected of havinga mitochondrial disorder. The mitochondrial disorder may be anymitochondrial disorder including, but not limited to, clinicalmanifestations such as seizures, strokes, optic atrophy, neuropathy,myopathy, cardiomyopathy, sensorineural hearing loss, diabetes mellitus.The sample taken from the individual may be any tissue sample, forexample, a blood sample. Magmas protein expression in the sample can beanalyzed by any method well known in the art including qualitative,quantitative mRNA analysis, quantitative protein analysis, e.g. Westernor dot blotting, or immunohistochemical analysis. The expression ofMagmas is thereafter compared to a normal control. If the expression ofMagmas is decreased in the sample from the individual compared to thecontrol, the individual is affected with a mitochondrial disorder. Thedecrease in expression may vary widely because the number of affectedmitochondria may vary due to known mosaicism phenomenon in mitochondrialdisorders. The decrease is considered significant if it is at leastabout 95% of the control, preferably at least about 50-70%, even morepreferably less than about 50% of the control sample. In the preferredembodiment, the analysis is an immunohistochemical analysis with anantibody directed against Magmas protein.

In another embodiment, the invention relates to a method of determiningthe severity of a mitochondrial disorder comprising the steps of takinga biological sample of an individual suspected of having a mitochondrialdisease, analyzing the Magmas protein expression in the sample,comparing the expression of the Magmas protein in the sample to a normalsample and/or comparing the expression of the Magmas protein in thesample to expression in a panel of samples consisting of tissue samplesrepresenting different degrees of severity of a mitochondrial disorder.The expression profile of the Magmas protein in the sample indicates thestage of mitochondrial disorder and provides prognostic information andguidance that can be used, for example in designing treatment options.For example, an individual with less than 50% of the normal Magmasexpression is considered more severely affected than an individual withabout 95% of the normal Magmas expression.

The invention further provides a method of diagnosing AML and/or ALL byidentifying truncation of the Magmas protein. More specifically, theinvention provides identification of one or more of the followingmutations:

MAKYLAQIIVMGVQVVGRVFARALRQEFAFARALRQEFAELZ, a frameshift deletion of 46codons at intron/exon site(SEQ ID NO: 5), results in 41 amino acidprotein);MAKYLAQIIVMGVQVVGRAFARALRQEFAASRFQPLRPQPPGGTADSQRVQ AEPZ (SEQ ID NO: 6),deletion of 14 codons results in 54 amino acid protein;MAKYLAQIIVMGVQVVGRAFARALRQEFAASRRHSRFSTCPSZ (SEQ ID NO: 7), frameshiftdeletion of 22 codons results in 44 amino acid protein;MAKYLAQIIVMGVQVVGRAFARALRQEFAASTPZ (SEQ ID NO: 8), a 174 bp insertresults in truncated 32 amino acid protein instead of 125 aa protein;andMAKYLAQIIVMGVQVVGRAFARALRQEFAASRAEADARGRAGHRSAAASNLSGLSLQEAQQKNYEHLFKVNDKSVGGSFYLQTKVVRAKERLDEELKIQAQ EDRKKGQMPHTZ (SEQ IDNO 9), a deletion of 13 amino acids, numbers 62-74 results in 112 aminoacid protein.

Most of the molecules known to be involved in GM-CSF mediated signaltransduction are also involved in IL-3 signaling. The a subunit of thereceptor complex determines ligand specificity and is required for IL-3and GM-CSF signal transduction. Studies with hybrid receptorsconstructed with the cytoplasmic domains of the murine IL-3 receptorshowed that only αβ heterodimers generated a mitogenic response [1,8].Homodimers of either α or β cytoplasmic domains were without activity.Mutagenesis of the cytoplasmic portion of the GM-CSF or IL-3 receptor asubunit suggested that specific regions of the receptor were importantfor proliferation, differentiation and survival [19-22].

While a few studies have described molecular differences between GM-CSFand IL-3 signal transduction pathways, most have shown them to beidentical. We have identified a novel mitochondrial associated proteinnamed Magmas whose role in GM-CSF activity in a murine myeloid cell linediffers from that of IL-3. Differential display was used to compare themRNA expression of PGMD1 cells cultured in GM-CSF to those cultured inIL-3. Using this approach we isolated a novel mitochondrial associatedgene, Magmas, whose message level was induced by GM-CSF. Consistent withits induction by GM-CSF, the promotor for Magmas contains an AP-1binding site which is known to be GM-CSF responsive [33], and a MZF-1[34) binding motif which is important in neutrophil development [35].

The mouse and human forms of Magmas protein are highly homologous.120/125 amino acids comprising the mature protein are identical, andthose that differ are generally conservative amino acid changes. Theprotein has a leader sequence which targets it to the mitochondria, asdemonstrated by immunohistochemistry and localization of GFP taggedprotein. Although sequences that target proteins to the mitochondriavary, the signal sequence of Magmas shares the characteristic amino acidcomposition of positively charged residues interspersed amonghydrophobic residues [36, 37]. The Magmas leader sequence isincompatible with those sequences that result in import into, orretention in the endoplasmic reticulum (includes golgi, lysosome,endosome and secretory vesicles), import into the nucleus, import intoperoxisomes, or attachment to membranes [38, 39].

The large 1st intron following the short 1st exon is not a featurecommon to many genes. This indicates that the intron has a regulatoryrole such as permitting the down stream methionine in the second exon toact as a translational initiation site. The detection of the Magmasdoublet on Western blot is compatible with initiation occurring at bothof these start sites. Initiation at the more distal site results in aprotein lacking a complete mitochondrial targeting sequence, which couldpotentially alter its localization and activity.

Results from Northern tissue dot blot showed that Magmas mRNA wasexpressed in all tissues in variable amounts. The highest levels wereobserved in heart, skeletal muscle and pituitary gland. mRNA levels infetal tissues were not as high as those in the corresponding adulttissues, implying that expression could be differentiation but notproliferation dependent. Significantly, many of the tissues with highMagmas mRNA levels by dot blot are not believed to express GM-CSFreceptor. Consequently, Magmas expression is also influenced by othertransduction pathways besides those regulated by GM-CSF.

GM-CSF induced Magmas mRNA expression in PGMD1 cells which had beengrowing in IL-3, we determined whether increased or reduced Magmasprotein levels would affect the ability of these cells to respond toeither growth factor. Full length sense and anti-sense cDNA wastransfected into PGMD1 cells and protein levels were measured by Westernblot. Compared to wild type cells, cells with overexpression of Magmashad similar proliferative rates and dose responsiveness in GM-CSF andIL-3. In contrast, cells with reduced Magmas proliferated poorly inGM-CSF. This effect was specific because the growth of these cells inIL-3 was comparable to the wild type and sense transfected cells (thelatter also serves as the vector control). However it is not known ifMagmas is required for growth of PGMD1 cells in IL-3 since we have notidentified any clones completely lacking expression of the protein.

Although not wishing to be bound by theory, there are several theoriesthat account for the activities of Magmas in PGMD1 cells. The first isthat Magmas affects the expression/activity of the GM-CSF receptor or isinvolved in pathways activated by this receptor. An example of a proteinhaving the latter characteristic is fps/fes, a cytoplasmic tyrosinekinase. In mice with homozygous fps/fes kinase inactivating mutations,growth factor stimulated phosphorylation of Stat 3 and Stat 5A wasimpaired for GM-CSF but not IL-3 [40]. It does not seem likely thatMagmas regulates receptor expression or interacts in the known signaltransduction pathways at this level because of its mitochondriallocation as well as its lack of homology to transcription factors or tocytoplasmic or membrane proteins.

Another possibility is that Magmas acts as an inhibitor of programmedcell death. Magmas has several features in common with Mcl-1, a bcl-2family member. Both are induced by GM-CSF [41], have a wide tissuedistribution [42], and co-localize with the mitochondrial compartment[43]. The lack of an apparent phenotype of PGMD1 cells with poor Magmasexpression grown in IL-3 may result from an analogous protein specificfor IL-3, or from the loss of responsive clones during post transfectionculture. However, most inhibitors of apoptosis are not growth factorspecific and Magmas does not have the BH4 domain or any other homologyto these proteins.

Magmas can also be an important regulator of mitochondrial activity andtherefore cell metabolism. A role in energy metabolism would explain thedivergent effect that reduced protein levels have on cells cultured inGM-CSF and IL-3. Although influenced by receptor expression, IL-3 andGM-CSF appear to differ in their ability to stimulate a respiratoryburst in neutrophils [44-46), and mononuclear cells [47-49]. The GM-CSFstimulated respiratory burst is characterized by the generation of toxicoxygen derivatives such as hydrogen peroxide and superoxide [50]. GM-CSFis sufficient for the respiratory burst in adherent neutrophils whilethose in suspension require an additional signal which experimentally isusually FMLP [46].

The respiratory burst is one component of the neutrophil activationprocess which also includes increased glucose transport [5]-53],increased glycolysis and Na⁺/H⁺ antiporter activity [54], enhancedphagocytosis and antibody mediated cytotoxicity [55]. It is likely thatGM-CSF and IL-3 have dissimilar activities on other aspects of theneutrophil activation process in addition to respiratory burst, but fewdefinitive experiments have been performed. Recently, using the yeast2-hybrid system, a protein has been identified which associates withGM-CSFα subunit but not the IL-3α subunit [56]. This protein named GRAP,affects glycogen accumulation in yeast and may be involved in some ofthe divergent effects that IL-3 and GM-CSF have on cellular metabolism.Similar to Magmas, GRAP was found to be expressed in all cell types.

Our data indicates that Magmas is involved in cell metabolism. Magmas isassociated with mitochondria and has high message levels inmetabolically active, non-proliferating, tissues such as muscle, adrenalgland, testis and liver. In PGMD1 cells with reduced Magmas expression,growth appears normal in IL-3 but not in GM-CSF. Induction of Magmasmessage by GM-CSF could be a compensatory response to the energyrequirement of the cell. Low Magmas levels should have similar effectson eosinophils grown either in GM-CSF or IL-3, since both of these HGFhave similar activating activities on these cells [57, 58].

Assays to further study Magmas exact role in mitochondrial activity andGM-CSF signaling in hematopoietic cells can look at mitochondrialmembrane permeability, electron transport and redox potentials, and itspotential interactions with proteins such as channel proteins, caspases,proteolytic inhibitors, and other members of growth factor signaltransduction pathways.

We have now discovered that the level of expression of Magmas isassociated with cancer. Examples of cancers associated with Magmas overexpression include, but are not limited to prostate cancer,neuroblastoma, Ewings sarcoma, osteosarcoma and leukemia. Prostatecarcinoma, one of the several malignancies known to express the GM-CSFreceptor or respond to GM-CSF, was examined for Magmas expression.Normal prostate stained slightly less strongly than benign hyperplasiawhich was less that the carcinoma samples. This was even true for thevarious histological grades found within a single tumor. Pathologicgrading of tumor samples revealed that Magmas expression closelycorrelated to the Gleason score. The level of Magmas expression alsoparallelled the level of GM-CSF receptor detected byimmunohistochemistry, suggesting a functional relationship.

In human prostate, several publications have shown little differencebetween the mitochondria in normal prostate tissue and those indifferentiated carcinoma (Kumamoto Medical J 20. In contrast, inundifferentiated carcinoma, mitochondria were reported to be morenumerous and had a more variable morphology. Unfortunately none of thesestudies normalized mitochondria content to cell size, making a directcomparison of these organelles very difficult. In hepatocellularcarcinoma, the differences in the number of mitochondria/cell wasrelated to the size of the cell.

The results of the electron microscopy showed that the mitochondriacontent did not significantly vary between normal and malignant prostatein agreement/disagreement with a previous study. EM was used toquantitate the mitochondrial content of the cells because of theuncertainly (that a particular protein is better) there are no bettermarkers available. Importantly, the amount Magmas/mitochondria wasincreased in the tumors compared to the normal prostate. Thisdemonstrates that Magmas is increased and does not simply reflect themitochondrial content of the cell.

The role of Magmas in the pathogenesis, progression or maintenance ofthe malignant phenotype in prostate cancer is unclear. Although thereare no published reports showing chromosomal translocations involving16p13.3 in prostate cancer, linkage analysis suggests that the entirearm of 16p could be involved in an increased susceptibility (Prostate45:106-114, 2000). Some cancer cells have been reported to haveincreased anaerobic metabolism compared to their normal counterparts. Itis possible that abnormal Magmas expression or activity may lead reducedaerobic metabolism by inhibiting the production of ATP. A potentialmechanism of action is suggested by co-immunoprecipitation studies andexperiments with the yeast two hybrid system demonstrating aninteraction between Magmas and the adenine nucleotide translocator 3(ANT). ANTs, found in the inner membrane of mitochondria, mediateADP/ATh exchange and have a role in oxidative phosphorylation andapoptosis.

The invention also provides a polymorphism (glutamine/lysine at position114 of the human gene) in the Magmas gene that is strongly associatedwith predisposition to prostate cancer. This polymorphism can beidentified in about 50% of prostate cancer patients whereas in normalpopulation, the prevalence is only about 3-4%. Therefore, thispolymorphism is useful in detecting individuals who may be predisposedto prostate cancer and require more frequent follow-up than individualswithout the polymorphism.

The invention further provides specific mutations in the Magmas. We haveidentified a point mutation in the Magmas gene in a patient with amitochondrial disorder having phenotypic features includingdevelopmental delay, hypotonia, ataxia, progressive limb weakness,exercise intolerance, myoclonus, failure to thrive and seizures. Thismutation decreases the expression of Magmas which assists in diagnosisas well as prognostics of the mitochondrial disorders. The pointmutation A->G results in a mutation E72G.

We have additionally identified several mutations in patients with AMLand ALL. These mutations include:

MAKYLAQIUVMGVQVVGRVFARALRQEFAFARALRQEFAELZ, a frameshift deletion of 46codons at intron/exon site(SEQ ID NO: 5), results in 41 amino acidprotein);MAKYLAQIVMGVQVVGRAFARALRQEFAASRFQPLRPQPPGGTADSQRVQ AEPZ (SEQ ID NO: 6),deletion of 14 codons results in 54 amino acid protein;MAKYLAQIIVMGVQVVGRAFARALRQEFAASRRHSRFSTCPSZ (SEQ ID NO: 7), frameshiftdeletion of 22 codons results in 44 amino acid protein;MAKYLAQIIVMGVQVVGRAFARALRQEFAASTPZ (SEQ ID NO: 8), a 174 bp insertresults in truncated 32 amino acid protein instead of 125 aa protein;andMAKYLAQIIVMGVQVVGRAFARALRQEFAASRAEADARGRAGHRSAAASNLSGLSLQEAQQKNYEHLFKVNDKSVGGSFYLQTKVVRAKERLDEELKIQAQ EDRKKGQMPHTZ (SEQ IDNO 9), a deletion of 13 amino acids, numbers 62-74 results in 112 aminoacid protein.

These mutations are useful in diagnosis of leukemia. For example, themutations can be screened for in a tissue sample, preferably in a bonemarrow sample obtained from an individual suspected of having leukemia.Detection of these mutations can be performed using methods well knownto one skilled in the art. Such methods include PCR-based methods,wherein nucleic acid, i.e. either DNA or RNA, in the sample is amplifiedusing primers flanking the mutation site. The difference in the size orthe sequence of the amplified nucleic acid is detected using gelelectrophoresis and/or nucleic acid sequencing and/or any other methodcapable of differentiating between size or sequence in the amplifiednucleic acid. Methods routinely used by one skilled in the art in suchapplication are presented, for example, in Sambrook and Russel,MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001), the entirety of whichis herein incorporated by reference.

EXAMPLE

Cell lines and culture. PGMD1, a murine factor dependent myeloid cellline [23], was maintained in hybridoma media (Sigma, St Louis, Mo.)supplemented with 15% FCS (Sigma) and murine IL-3 (10 ng/ml; Preprotech,Princeton N.J., or Sigma). Magmas sense and antisense DNA in the vectorpREP4 (Invitrogen, San Diego, Calif.) was transfected into PGMD1 cellsby electroporation (260 volts, 960 μP; Gene Pulser, BioRad, Hercules,Calif.). Immediately after electroporation, the cells were plated inmicrowell dishes, and two days later were selected by culture inhygromycin (375 units/ml, Calbiochem, San Diego, Calif.). Statisticallymost wells contained a single colony. Magmas-green fluorescent proteinfusion protein (linked 5′ Magmas-GFP3′) and green fluorescent protein(GFP) (EGFP, Clontech, Palo Alto, Calif.) alone were also expressed inPGMD1 cells using pREP4 and identical techniques.

Differential Display. PGMD1 cells exponentially proliferating in IL-3were washed 3 times in warm GM-CSF containing media and then cultured inmurine GM-CSF (40 ng/ml; gift from Genetics Institute, Cambridge Mass.).RNA was prepared using RNAzol B (Biotecx, Houston, Tex.) andMessageClean (GenHunter, Nashville Tenn.) from cells grown in IL-3 (T=0hr) or in GM-CSF for 6, 12, or 24 hours. cDNA reverse transcribed fromthe polyA mRNA was amplified by PCR using differential display primersand reagents (GenHunter) and ³⁵S DATP (NEN, Boston, Mass.). Theresulting products were subjected to polyacrylamide gel electrophoresisand the results from each time point were analyzed afterautoradiography. Each reaction was performed on RNA isolated from threeindependent experiments. Fragments which showed consistent changes overthe three experiments were excised from the gel, and cloned into thepCR-TRAP vector (GenHunter). The sequence of each fragment wasdetermined by automated DNA sequencing using standard techniques.

Northern Hybridization. Total RNA was prepared (Rneasy, Qiagen,Valencia, Calif.) from PGMD1 cells grown in murine IL-3 or murine GM-CSF(PeproTech) for the time periods indicated. 10 μg total RNA per lane wasfractionated on a 1% agarose formaldehyde gel at 75 volts for 3 hours[24]. After overnight upward transfer in 20× saline sodium citrate (SSC;150 mM NaCl/15 mM Sodium Citrate) to Duralon-UV membrane (Stratagene,LaJolla, Calif.) the RNA was crosslinked (UV Stratalinker 2400) andstained with methylene blue to confirm uniformity of loading andtransfer. Magmas and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)probes were radiolabeled with ³²P dCTP NEN, Boston, Mass.) using randomprimers (HighPrime, Roche, Indianapolis, Ind.) and then column purified(ProbeQuant G-50, Pharmacia, Piscataway, N.J.). After hybridization(QuickHyb, Stratagene, La Jolla, Calif.) overnight at 68° C., the blotswere washed twice in 2×SSC/0.1% sodium dodecyl sulfate (SDS) and twicein 0.1×SSC/0.1% SDS, and exposed to film. Northern blots were performedon RNA isolated from three independent experiments.

A multiple tissue array dot blot was probed with a random primer ³²PdCTP labeled Magmas probe or a ubiquitin control probe with ExpressHybhybridization solution block, according to manufacturer's directions(Human MTE Array, Clontech). After a 3 day exposure on a phosphoimagerplate the data was analyzed using ImageQuant 1.2 on a Storm 860Phosphoimager (Molecular Dynamics, Sunnydale, Calif.). The entirelisting of mRNA samples on the membrane can be found atwww.clontech.com/products/catalog01/Sec1/imagesSec1/p50fig1600.gif.

Cloning of cDNA and genomic DNA. A cDNA library was prepared from mRNAisolated from PGMD1 cells cultured for 48 hours in GM-CSF. 10 ng of cDNAwas converted to double stranded DNA by nick translation and ligatedinto the pSport vector (Superscript plasmid system, Life Technologies,GIBCO BRL, Gaithersburg, Md.). An oligonucleotide probe(gagcgtccgccatggccaagta) [SEQ ID NO: 1] derived from the differentialdisplay fragment sequence was used to isolate the murine cDNA cloneusing the GeneTrapper cDNA positive selection system (GIBCO BRL). Themethod consists of degradation of one strand of the library plasmidswith exonuclease III; solution hybridization with the specificbiotinylated oligonucleotide probe; capture of positive clones onstreptavidin beads; and priming and repair of the plasmid to doublestrandedness. Selected clones were screened for inclusion of thedifferential display fragment sequence. The translational start site,leader sequence and regions of interest were identified by SMART [25,26], (Simple Modular Architecture Research Tool) atwww.smart.embl-heidelberg.de/. The GenBank accession number assigned tomurine Magmas is AF349454.

Human Magmas cDNA and genomic DNA were cloned from peripheral blood cellby PCR using primers designed from comparing the mouse cDNA and a humancosmid clone sequence found in GenBank™-5′ and 3′ untranslated regionsof human T2c12 cDNA were cloned by the RACE method [27, 28], using aseries of primers selected by the Oligo program (Molecular BiologyInsights, Cascada, Colo.). The human Magmas GenBank accession number isAF349455. Transcription factor binding sites were identified by theTFSEARCH program (www.rwcp.orjp/papia) utilizing the TRANSFAC database[29].

Polyclonal Antibody. Mixtures of human and murine peptides comprised ofthe 19 amino-terminal (N-terminal) terminal or 20 carboxy-terminal(C-terminal) amino acids were synthesized and coupled to KLH (DanaFarber Cancer Institute Core Facility, Boston, Mass.) and inoculatedinto rabbits (Covance, Richmond, Calif.). The antibodies were generatedusing fragments having the N-terminal amino acid sequence:

DEEL(R/K)IQAQEDREKGQ(K/L)P(K/H)T [SEQ ID NO: 2]

and a C-terminal amino acid sequence:

RALRQEFAAS(Q/R)AAADARGRC. [SEQ ID NO: 3]

Antisera and the preimmune sera were screened against lysates frombacterial clones expressing murine Magmas, human Magmas or a nonspecific similar sized control protein by Western blot. Forimmunohistochemistry, the immune, preimmune, and control (from rabbitsinjected with a nonreactive KLH coupled peptide) sera were purified on aHiTrap protein G column (Amersham Pharmacia Biotech, Piscataway, N.J.).The concentration of the eluted protein was measured by the Bradfordmethod (BioRad) using immunoglobulin as the protein standard.

Immunohistochemistry. Cytospin prepared slides were blocked in PBScontaining 1.5% BSA and 2% non fat dry milk. The slides were thenincubated in purified immune and control antibody (10 ng/ml) in PBS/BSAfor 1 hr at 37° C., washed and incubated with FITC conjugatedanti-rabbit antibody. After 3 rinses, the slides were counterstainedwith Vectorshield with propidium iodide (Vector Laboratories, BurlingameCalif.), and examined under fluorescent microscopy. Subcellularfractionation of mitochondria was achieved by dounce homogenization anddifferential separation through 0.25 M sucrose, using the succinatedehydrogenase assay as the marker [30].

Human prostate tissue was obtained from surgical prostatectomy samplesor archival blocks with the approval of the Institutional Review Boardsof Children's Hospital Medical Center and the University of CincinnatiMedical School. The prostate samples for morphology and lightimmunohistochemistry were fixed in paraformaldehyde, dehydrated inethanol containing solutions, embedded in paraffin and sectioned. Slidesfrom each sample were stained with H and E and graded according to theGleason classification.

Magmas expression was determined by incubating the samples in protein Gpurified polyclonal anti-Magmas antibody or control antibody (10 ng/ml)followed by biotinylated goat anti-rabbit IgG (Vector Laboratories,Burlingame, Calif.) and streptavidin congugated horse radish peroxidase(Dako, Carpinteria, Calif.). The slides were then incubated withdiaminobenzidine (DAB) solution (Dako). Similarly to detect the GM-CSFreceptor expression, sectioned tissues were sequentially incubated inanti-GM-CSF receptor a or isotype control antibody (both at 10 μg/ml),horse radish peroxidase conjugated anti-rabbit IgG and DAB, (VentanaMedical Systems, Tucson, Ariz.). The results of histological grading andthe scoring were done independently and blinded.

Magmas expression was determined by incubating the samples withanti-Magmas antibody or control antibody followed by biotinylated goatanti-rabbit IgG. Similarly, human tissues were sequentially incubated inanti-GM-CSF receptqra subunit antibody (Alpha Diagnostics, San Antino,Tex.) or isotype control antibody (Sigma), anti-rabbit IgG-horse radishperoxidase and All slides were counter-stained with Mayers hematoxylin.

The electron microscopy samples processed as previously described.Normal and malignant prostate tissue were fixed in 4%paraformaldehyde/2.5% glutaraldehyde followed by fixation in 2% osmiumtetroxide (Electron Microscopy Sciences, Ft. Washington, Pa.) reducedwith 1.5% potassium ferrocyanide both in Hank's Balanced Salt Solutioncontaining 5% sucrose. After dehydration, and embedding in Eponate (TedPella, Redding, Calif.), the tissue was sectioned and placed on meshnickel grids (Electron Microscopy Sciences) [31]. Next the grids wereincubated with anti-Magmas antibody or control antibody followed bybiotinylated goat anti-rabbit IgG (KPL Laboratories, Gaithersburg, Md.),and streptavidin:5 nm gold colloid [32]. The samples were then stainedwith 2% aqueous uranyl acetate (Electron Microscopy Sciences) and viewedin a JEOL 100CX II electron microscope. The distribution and location ofthe gold grains/micron² was determined by stereologic analysis using thepoint counting method [33]. The samples were scored as the number ofgrains/micron² of mitochondria and the of area of mitochondria dividedby the total area of sample.

Western Blot. Equal numbers of PGMD1 cells and cells expressing GFP or aMagmas-GFP fusion protein were solubilized in Laemmli sample buffer andthe proteins were separated by SDS-PAGE. The proteins were transferredto a nitrocellulose membrane (TransBlot Transfer Medium, Biorad) in aTris/Glycine-20% methanol solution overnight (100 volt-hr) at 4° C.[31]. Membranes were rinsed, blocked and then incubated with anti-Magmasantisera ( 1/500). After washing, the membrane was immersed for 30minutes with peroxidase-labeled secondary antibody ( 1/10,000), washedfour times with large volumes of buffer containing 50 mM Tris pH 7.5/150mM NaCl/0.1% Tween 20 (TBST), for 15 minutes each, developed in luminolsubstrate and exposed to film (BM Chemiluminescence Western BlottingKit, Roche Molecular Biochemicals, Indianapolis, Ind.). The blot wasthen stripped and similarly reprobed with a mixture containing preimmunesera and rabbit anti-GFP antibody. Kaleidoscope Prestained Standards(BioRad) were used for molecular weight determinations. Relative loadingof the lanes was confirmed by Ponceau S (Sigma) staining of the membrane[32].

Proliferation assay. After washing 3 times, PGMD1 cells were plated at6000 cells/well and cultured at 37° C. for 72 hrs in 160 μl mediacontaining 15% FCS without factor, or with varying concentrations ofIL-3 or GM-CSF as indicated. The cells were then cultured for anadditional 4 hours with 1 μCi/well of ³H thymidine ZEN), harvested usinga FilterMate Harvester and counted on a TopCount MicroplateScintillation Counter (Packard, Meriden, Conn.). Each time point is themean of three wells, and each experiment was performed twice.

Expression of Magmas message in tumor and normal prostate tissue. Amultiple tissue array dot blot consisting of RNA derived cDNA was usedto determine Magmas mRNA expression from malignant and normal prostatetissue from the same patient (Matched Tumor/Normal Expression Array, BDBiosciences Clontech). The cDNA on the membrane, which accuratelyreflects the relative abundance of mRNA, have been normalized by themanufacturer to ubiquitin, ribosomal protein S9 and 23 kD highly basicprotein. The membrane was probed with a random primer ³²P dCTP labeledMagmas probe or a GAPDH control probe with ExpressHyb hybridizationsolution block, according to manufacturer's directions (Human MTE Array,Clontech). After a 3 day exposure on a phosphoimager plate the data wasanalyzed using ImageQuant 1.2 on a Storm 860 Phosphoimager (MolecularDynamics, Sunnydale, Calif.).

Differential expression of PGMD1 mRNA from cells cultured in I-3 andGM-CSF. The growth factor dependent cell line PGMD1 was used to identifygenes that were differentially responsive to IL-3 and GM-CSF. RNAisolated from exponentially proliferating cells grown in IL-3 (T=0) orcells cultured for 6, 12, and 24 hours in GM-CSF, was analyzed bydifferential display. After amplification by PCR the products weresubjected to polyacrylamide gel electrophoresis and the PCR fragmentscompared at the different time points. The increased number of PCRfragments observed when the cells were grown in GM-CSF was typical formany of the primer sets used during the amplification step. Thirty fivePCR fragments showing differential expression at the various time pointswere cut out of the gel and cloned. An autoradiograph of thedifferential display gel containing the PCR product from which afragment of Magmas was isolated is shown in FIG. 1. To the left of thestar is the band which encodes a fragment of Magmas cDNA.

Message levels are regulated by GM-CSF. To confirm that the cDNAfragments identified by differential display had variable levels of mRNAexpression when the cells were cultured in IL-3 or GM-CSF, Northern blotanalysis was performed using probes derived from the cloned fragments.Fourteen of the 35 fragments selected for further analysis were found tohave variable expression over time. As shown in FIG. 2A, one of thegenes which had a single transcript size of approximately 500 kb wasweakly expressed in PGMD1 cells grown in IL-3 (T=0). After 6 hours ofculture in GM-CSF, the amount of transcript was increased. Transcriptlevel continued to rise to a maximum of approximately 5-6 fold at 24hours of GM-CSF exposure. At T=48 and 96 hr the transcript level waslower than that seen at T=24, however it was still higher than theamount detected in cells growing in IL-3. The blot was then stripped andre-probed with DNA complimentary to GAPDH (FIG. 2B) as a control forsample loading and transfer. Consistent with methylene blue stainingprior to hybridization, the GAPDH signal was approximately equal inevery lane. These results demonstrate that GM-CSF was able to induceMagmas transcript levels over those observed when PGMD1 cells are grownin IL-3.

Gene and Protein Structure. A murine cDNA library derived from PGMD1cells grown in GM-CSF was used to isolate the Magmas gene. Clones wereselected from the library using an oligonucleotide probe based on thedifferential display fragment sequence. The cDNA isolated was 536 bp inlength and contained the original differential display DNA fragment atthe 3′ end (FIG. 3A). The clones consisted of a 78 bp 5′ untranslatedregion, a 375 bp open reading frame and an 83 bp 3′ non coding segment.

After characterizing the murine cDNA, the Genbank database was searchedfor a human equivalent. BLAST comparison to the murine Magmas sequenceyielded a single match with 87% homology to regions of a human cosmidclone RT140 (AC004789) located on chromosome 16p13.3. Despite the highdegree of homology to the murine cDNA sequence, the equivalenttranslational start site for the human homolog was not readily apparentfrom examination of the genomic sequence.

To determine the sequence of the N terminal portion of the human gene,we used internal primers to amplify the 5′ and 3′ untranslated regionsof human cDNA by the RACE method. The sequence of human cDNA wasidentical to discontinuous regions in the human genomic clone. Theintron-exon boundaries conform to the GT/AG rule for the splice donorand acceptor sites. There is a typical translational start siteconsisting of a Kozak sequence and fMet initiation codon. Binding sitesfor transcriptional factors AP-1, GATA1, GATA2, GATA3, Ik-1, AML-1a,MZF-1 and c-Ets are located upstream of the transcriptional start site.Based upon the human cDNA sequence and genomic sequence the human geneis comprised of 5 exons and 4 introns and contains 12,975 bases (FIG.3B). Interestingly, the first exon consists of just the 5′ untranslatedregion and the fMet codon and is separated from the second exon by a 9.9kb intron.

The protein predicted for Magmas consists of 125 amino acids including a21 residue leader sequence identified by the SMART protein analysisprogram (FIG. 3B). The leader sequence consists of many hydrophobicamino acids, a few basic amino acids and no acidic residues, and is onlyconsistent with sequences that target proteins to the mitochondria.Although the protein did not contain any known functional domains therewere two regions of low compositional complexity identified at the Nterminus (aa 23-41 and 45-56) and a region having some similarities to aDnaJ domain (aa 57-110). In the absence of any postranslationalmodification, a protein initiated at the first start site would have anexpected molecular weight of 13.7 kd. A comparison of the coding regionsof the human and murine genes showed amino acid differences only atpositions 32, 41, 110, 122 and 124 (FIG. 3A). A polymorphism resultingin a change from glutamine to lysine at position 114 has also beendetected in the human sequence.

The molecular weight of the native protein was estimated by Western blotanalysis (FIG. 4). Lysates from wild type PGMD1 cells or controls (cellstransfected with constructs encoding a Magmas-GFP fusion protein or GFPalone) were subjected to polyacrylamide gel electrophoresis, transferredto nitrocellulose and blotted with antibody against Magmas (lanes 1-3).After incubation with peroxidase coupled anti-rabbit antibody andaddition of chemiluminescent reagent, the blot was exposed to film. Inlane 1 (wild type cells), a major fragment having a molecular weight ofapproximately 13 kD was observed. This correlates with the molecularweight predicted from the amino acid sequence. Cells transfected withthe Magmas-GFP fusion protein (lane 2) had two major fragments. Thelower corresponds to the endogenous Magmas protein seen in lane 1 whilethe upper has the expected molecular weight of the fusion protein. Thecells containing the construct encoding only GFP were similar to thewild type cell sample when blotted with the anti-Magmas antisera (lane3).

The blot was then stripped and re-probed with a mixture of controlpreimmune sera and antibody against GFP (lanes 4-6). Under theseconditions the lower band was no longer present in any of the lanes.This demonstrates that anti-Magmas serum specifically recognizes the 13kD protein. When the cells transfected with the fusion protein constructwere blotted with the anti-GFP/preimmune sera mixture (lane 5) a highmolecular weight band identical to the one in lane 2 was observed. Thistogether with the result seen in lane 2 conclusively proves that the 13kD protein seen in lanes 1-3 is Magmas. In lane 6, (cells transfectedwith GFP alone) a single band having the expected molecular weight ofGFP (27 kd) was visualized, confirming the specificity of the anti-GFPantibody.

When PGMD1 cell lysates were separated on a higher resolutionpolyacrylamide gel the Magmas protein fragment was shown to consist of adoublet having molecular weights of 11,460 and 12,820 daltons (lane 7)on Western blot. The former corresponds very favorably to the molecularweight of the Magmas with a cleaved signal peptide (11,530 daltons) andthe latter to a Magmas protein initiated at the distal methionineresidue (12,650 daltons). The difference in size appears too small forthe fragments to represent the protein with or without the leadersequence. Occasionally a fragment of approximately 25 kD has been seenon Western blot of whole cell lysate. The blot used in FIG. 4 providesthe most prominent example of this fragment (lane 2, middle band).Unlike Magmas, this band is not immunoprecipitated by the polyclonalantibody to Magmas and does not bind to an anti-Magmas immunoaffinitycolumn (data not shown). This demonstrates that the responsible epitopeis only recognized when the protein is in a denatured state. The exactnature of the 25 kd band is unknown but the possibilities include aprotein derived from another transcript, a posttranslationalmodification of Magmas, or a protein containing a cross-reactingepitope.

Expression of Magmas mRNA in tissues. A Northern dot blot containing RNAderived from a variety of human tissues was used to determine the tissuedistribution of Magmas. To get the best approximation of relative mRNAlevels, the amount of poly A⁺ mRNA loaded for each tissue was normalizedto 8 different housekeeping genes by the manufacturer. A quantitativemeasure of Magmas mRNA expression was obtained by phosphoimageranalysis. The mean signal volume for all of the human tissues was 6644with a standard deviation (n-1) of 6377. Every tissue on the blot had adetectable amount of message. The volumes are available upon request.

Tissues were grouped into categories of relative Magmas mRNA expressionaccording to the level of standard deviation from the mean volume (Table1). The highest mRNA levels were present in the heart and skeletalmuscle. Intermediate levels of mRNA were found in the adrenal gland, thecaudate nucleus and putamen region of the brain, the pituitary gland andthe testes. Somewhat surprisingly, the mRNA levels in the spleen (5664),bone marrow (5983) and peripheral blood leukocytes (5775) were all nearthe mean value for all tissues. mRNA expression in fetal tissues such asheart (3739) and liver (5070) were at least 3 fold lower than those ofthe corresponding adult tissues (shown in Table 1) demonstrating thatincreased transcript levels are not a marker for cellular proliferation.There was no signal in the yeast mRNA. Normalization of the tissuevolumes to a membrane hybridized with an ubiquitin probe reduces thevalues of the cardiac tissues and skeletal muscle relative to the othertissues (data not shown), but they still remained in the high expressioncategory.

TABLE 1 Human tissues with high expression of Magmas mRNA. Tissue volumeTissue volume >Two standard deviations from the mean right ventricle28329 interventricular septum 23098 apex of heart 26453 left ventricle20355 skeletal muscle 23393 >One standard deviation from the meanpituitary 18043 ileum 14003 left atrium 15512 caudate nucleus 13968liver 15445 putamen 13578 heart 14988 right atrium 13531 adrenal gland14052 testes 13304 Signal volume quantified by phosphoimager. Meanvolume = 6644. Standard deviation (n − 1) = 6377. Representative of twoexperiments performed on the same membrane.

Magmas localizes to the mitochondria. Immunohistochemical staining ofPGMD1 cells was used to confirm the expected mitochondria location ofMagmas predicted from the amino acid composition of its leader sequence.In FIGS. 5A and 5B, cells were incubated with anti-magmas antibody (FIG.5A) or preimmune antibody (FIG. 5B), followed by fluorescein labelledgoat anti-rabbit antibody. To visualize the nuclei the cells werecounterstained red with propidium iodide. FIG. 5A shows the fluorescentpattern of anti-Magmas antibody to be distributed in a punctate patternin the cytoplasm. None of the fluorescence signal is found in thenucleus. This pattern is consistent with Magmas having mitochondriallocation. PDGM1 cells similarly treated with control antibody (FIG. 5B)did not have any detectable fluorescein signal.

To further demonstrate the cellular location of Magmas, its cDNA waslinked at the 3′ end to DNA encoding GFP, and transfected into PGMD1cells. A vector containing GFP alone was used as control. FIG. 5C showsthe fluorescent signal in the cells containing Magmas-GFP in a punctatecytoplasmic distribution, identical to that observed in FIG. 5A. Controlcells expressing GFP had a fluorescence signal uniformly distributedthroughout the cell (FIG. 5D). Examination of mitochondrial preparationsby fluorescent microscopy showed that the samples derived from cellstransfected with Magmas-GFP were intensely stained (panel E), whilemitochondrial preparations from the cells transfected with GFP alone hadlittle signal (panel F).

Electron microscopy (EM) studies were then performed to verify thesubcellular fractionation experiment showing that Magmas is found in themitochondrial compartment. Tissue sections of human prostate wereincubated with Magmas antibody or control antibody, followed bybiotinylated goat anti-rabbit Ig and streptavidin:gold colloid.Representative electron micrographs of the results are shown in FIG. 6.In samples incubated with the anti-Magmas antibody (panel A), manyclusters of electron dense gold particles are found in the mitochondria(arrowheads) and in the rough endoplasmic reticulum/ribosomalcompartment (arrows). No particles are observed elsewhere in themicrograph shown. In contrast few gold grains are observed in prostatesamples incubated with the control antibody (panel B). The arrowindicates the position of two grains located near the ribosomalcompartment.

Greater than 100 of these electron micrographs were used to accuratelyquantify the distribution of gold grains per unit area. In the samplesstained with anti-Magmas antibody, gold particles were found to localizeto the mitochondria compartment (85.93+/−47.51; meanparticles+/−standard deviation per micron²) or the rough endoplasmicreticulum/ribosome compartment (80.94+/−52.46) in comparison to the“other” compartment (14.57+/−8.21). The labeling of the mitochondria andribosomal compartments were not significantly different (p>0.05) fromeach other, but each were statistically greater than the “other”compartment (p<0.001). A similar evaluation of the micrographs ofsamples incubated with control antibody showed there were 3.11+/−3.82mean+/−SD particles/micron² in the mitochondria, 3.22+/−3.27particles/micron² in the endoplasmic reticulum/ribosome compartment and15.89+/−12.29 particles/micron² in the “other” compartment. Unlike theresults with the anti-Magmas antibody, chi square analysis shows thatthe gold grains in the antibody control are randomly distributed(p=0.15) throughout the cell.

These EM experiments show that Magmas is found only in the mitochondriaor in the closely associated ribosomal compartment where translationoccurs. Thus it is likely that the upper band of the doublet in FIG. 4,lane 7 represents ribosomal associated Magmas containing its leadersequence, and the lower band the processed mature form of Magmas foundin the mitochondria

Effect of Magmas expression on IL-3 and GM-CSF stimulated proliferation.Full length sense and antisense cDNA constructs of Magmas were ligatedinto the pREP4 vector under the control of a constitutively active CMVpromotor and electroporated into PGMD1 cells. Cells were plated inmicrowell dishes and selected with hygromycin. The Magmas protein levelsof the resulting transfectants were then determined by Western blot(FIG. 7A). Transfected cells with increased levels of Magmas are shownin lanes 1 and 2. Intermediate Magmas expression was observed in thewild type, untransfected cells (lane 3), and lanes 4 and 5 weretransfected cells exhibiting reduced expression. We were not able tofind any antisense transfectants that had undetectable Magmasexpression.

The transfected and wild type cells from FIG. 6A were then tested fortheir ability to proliferate in IL-3 and GM-CSF as measured by³H-thymidine incorporation. The cells with increased or reduced Magmasexpression grew similarly to the wild type, non-transfected cells in thepresence of IL-3 (FIG. 7B, left panel). When cultured in GM-CSF, cellswith increased expression also proliferated like the wild type cells(FIG. 6B, right panel). In contrast, GM-CSF dependent proliferation wassignificantly impaired in cells with low levels of Magmas protein.Examination of these cells showed that reduced viability was responsiblefor some of the decrease in ³H-thymidine incorporation.

Magmas message levels are elevated in prostate cancer. A blot containingmatched samples of cDNA derived from normal and malignant prostatetissue obtained from the same patient was probed with an ³²P labelledolignucleotide to Magmas cDNA. The results show that, the level ofMagmas message is 3.5 times higher in the tumor than in normal prostatetissue for 3 cases. Even though the sample loading was normalized to 3housekeeping genes by the manufacturer, the blot was stripped andre-probed for GAPDH expression (FIG. 7B) as an independent control forthe reverse transcriptase reaction and sample loading.

Increased expression of Magmas and GM-CSF receptor in prostate tumors.Prostate samples obtained from surgical specimens were examined forMagmas expression by immunohistochemistry. High grade prostate cancerwas stained with Hematoxylin-Eosin, anti-Magmas antibody and anti-GM-CSFreceptor antibody. Malignant areas of the prostate sample showedsignificant staining with both Magmas and the GM-CSF receptor, whilelittle staining was observed in the normal areas.

Magmas protein expression correlates to grade. Prostate samples from 13patients were stained with an antibody against Magmas. The set includedthe full extent of prostate specimens from normal and benign hyperplasiato high grade metastatic prostate cancer. Normal prostate wasessentially negative for Magmas expression. Benign hyperplasia wasobserved to have slightly elevated expression. For each carcinomasample, the intensity of the staining was directly proportional to thegrade of severity, the most severe high grade metastatic prostate cancersample having the most intensive staining.

Magmas expression in prostate cancer is independent of the mitochondrialcontent of the cell. We have shown that Magmas message and protein havebeen shown to be higher in prostate cancer than in normal prostatetissue. This increase may result from higher Magmas expression in eachmitochondria or more mitochondria cell. To accurately distinguishbetween these two possibilities, electron microscopy was used toquantitate the volume of mitochondria/cell and the relative expressionof Magmas/mitochondria based on immunohistochemical staining. The numberof mitochondria per cell in high grade prostate cancer and normalprostate tissue was not found to be statistically different. Incontrast, the amount of Magmas expression in the mitochondriacompartment (86 grains per micron² of mitochondria) was much higher thanthat found in the mitochondria of the normal prostate tissue, 9 grainsper micron² of mitochondria.

Tissue dot blot and immunohistochemistry on murine embryos from E 6.5through adult tissues. In order to gain insight about the role of Magmasin vivo, mRNA levels and protein expression were examined in murineembryos and adult tissues. A survey of Magmas mRNA in various murinetissues was performed using a tissue dot blot. The samples on the blotwere normalized to eight housekeeping genes by the manufacturer. Theautoradiograph of the hybridization of ³²P labeled Magmas probe detectedMagmas mRNA in all of the adult murine tissues as well as in the 4 wholeembryo samples represented on the blot. The mean signal intensity forall adult tissue was 167,415 with a standard deviation (SD; σn-1) of183,319. Higher levels of Magmas mRNA were found in testes (937,420; >3SD from mean) and heart (491,236; >1SD from the mean). Liver (119,275),smooth muscle (107342) and pancreas (95,588) had the lowest signals, butthey did not vary from the mean by more than 1 SD. The values from wholemouse embryos showed increasing signal intensity with age from day 11 today 17, but the highest value was on day 7 embryo sample. The signalsobserved for the negative controls (yeast total RNA, yeast tRNA, E colirRNA, E coli DNA, poly r(A), and repetitive DNA sequences) were allsimilar to the background. In a separate Northern blot analysis, theamount of Magmas message in placenta was similar to that of kidney.

High level of protein and mRNA levels in testes suggests that abnormalMagmas could be involved in fertility problems.

Expression of Magmas protein during murine development. Proteinexpression during murine development was examined at day 6.5, 10, 12.5,14.5, 15.5 and day 18 by immunohistochemistry using anti-Magmasantibody. The amount of protein expressed was reflected by the intensityof brown precipitate resulting from the peroxidase reaction.

An intermediate amount of staining was observed in the proximal anddistal endoderm including Reichert's membrane, ectoplacental cone, theembryonic ectoderm, and the decidua in the sections from a day 6.5embryo. The section through placenta showed strong staining in the yolksac epithelial lining, as well as the parietal endoderm and surroundingdecidual cells.

In the day 12.5 embryonic heart strong staining occurred in the myocytesin the atria as well as the muscle cells in the region of theatrioventricular canal. No significant staining was observed in thestromal cells of the endocardial cushions. Enhanced staining wasobserved in a section through a spinal cord region of the day 12.5embryo in the notochord, the spinal ganglia and the somites, which giverise to the skin and skeletal elements.

In the day 14.5 embryo intense staining was observed in the epitheliallining of the choroid plexus of the developing brain. The primitiveneuroectodermal layer also showed weaker staining. The stroma underlyingthe epithelial cells in the choroid plexus were negative. There was nosignificant staining observed in this region of the brain at day 12.5.There is strong staining in the neural cells throughout the ganglia inthe section through the cervical ganglion at day 14.5. In contrast, theconnective tissue surrounding the ganglia showed no significantstaining. Also, in the day 14.5 embryo, strong staining was observed inthe developing muscle layers. In addition, Magmas expression was alsoseen in the cartilage of the developing ribs as well as in the adjacentspinal ganglia adjacent to the muscle layers. The spinal ganglion cellsstained less than the cervical, because they are developmentally moreimmature. There was strong staining in the epithelial lining of theintestinal tract as well as weaker staining in the developing muscularwall of the intestinal tract in a section through the developingintestinal tract of the day 14.5 embryo. No significant staining wasobserved in the developing diaphragm, however the liver was stronglypositive.

In the day 18 embryo, besides the staining of the tissues alreadydiscussed there was very strong staining in the salivary glandepithelium although the surrounding connective tissue was negative. Thenasal mucosal epithelium in the underlying submucosal glands, which weremorphologically identifiable at day 17, also showed strong expressionduring this stage of development. Magmas staining for many of the othertissues at Day 18 were similar to that observed at earlier stages. Themost striking exceptions occurred in skeletal and cardiac muscle, liverand bronchioles, where staining was reduced to negligible levels.

As shown in Table 2, the level of Magmas expression varied considerablyduring embryonic development. For example, Magmas expression waselevated in skeletal muscle, beginning at the inception ofmorphologically identifiable fibrils. The high level continues untilsometime between day 15 and day 18 when Magmas is barely detectible.Liver, cardiac muscle, and bronchioles also had decreased Magmasexpression in the day 18 sections. In contrast, staining of renalstructures occurred relatively late (weak on day 15.5) and was higheston day 18.

Magmas protein expression in adult tissues Tissues from the adult mouseincluding brain, spinal cord, liver, spleen, pancreas, small bowel,heart, lung, kidney, ovary, uterus, and testes were evaluated for theexpression of Magmas by immunohistochemistry. The amount of expressionin adult tissues was reflective of the continuously high levels ofMagmas protein observed throughout muscle development beginning with thefirst appearance of immature muscle in the embryonic tissue. Cardiacmuscle fibers showed a variable reaction pattern with many stronglypositive fibers in contrast to clusters of numerous negative fibersadjacent to the positive fibers. Also, even in the positive stainingfibers there was variability within the fiber of the staining intensity,with some fibers showing more intense staining at the periphery of thecell or occasionally in a perinuclear distribution. Skeletal musclefibers also showed strong positive staining. Most of the muscle cellswere strongly positive, and in the skeletal muscle fibers the stainingwas associated with the filaments and z-bands. Cardiac muscle showedcompartmentalization of the staining pattern in a coarse, granularpattern, usually with a perinuclear distribution.

Other organs, such as the kidney, pancreas, and intestine, showedpositive staining for Magmas. Liver showed diffuse cytoplasmic stainingin most of hepatocytes and no significant staining of the bile ductepithelium. The kidney section showed intense staining in the perirenalfat cells. Prominent expression was also observed in the proximaltubules in the renal cortex. In contrast to the cortex, the renalmedulla showed little evidence of staining of the tubular epithelialcells. The glomeruli were also negative. The pancreas had diffusepositive cytoplasmic staining in the acinar cells. The intestinal tractshowed positive staining in the mucosa epithelial lining of the smallbowel.

The reproductive tract showed extensive evidence of Magmas expression.In the mouse, the testes were among the tissues having high levels ofMagmas mRNA by Northern blot. Using antibody on adult testes strongstaining in the interstitial Leydig cells as well as positive stainingin the spermatocytes and spermatids was demonstrated. The epitheliumlining the epididymis was also strongly positive. In the femalereproductive organs, the ovary, unlike the testes, did not show muchMagmas expression. Both the follicle and the perifollicular cellsstained negative. The only cell populations in the ovarian tissue thatwere positive were the fibrous tissue and blood vessels in the ovarianstroma. The endometrial mucosal cells lining the of murine uterus samplewere weakly positive.

The brain shows strong Magmas antibody staining in the Purkinje cells ofthe cerebellum. The granular cells in the white matter of the cerebellarcortex do not express significant amounts of Magmas. In the cerebralcortex of the adult murine brain, high levels of Magmas expression wasobserved in neuronal cells, although the glial cells were generallynegative or showed weak staining (data not shown). In contrast to thedeveloping spinal cord, the adult spinal cord did not show significantstaining. Minimal expression of Magmas was observed in other organs suchas the lung, spleen and thymus.

As one example of a negative control for the anti-Magmas antibody wasthe section containing choroid plexus incubated with preimmune serum asthe primary reagent. Each the panel had a matching negative control,which showed minimal to no background staining. Inclusion of a 10 foldmolar excess of uncoupled immunizing peptide with the anti-Magmasantibody eliminates Magmas staining on testes and prostate sectionsfurther demonstrating the specificity of the antibody.

Discussion

Magmas protein was detected as early as the day 6.5 embryo and was foundin each of the three germ lineages. Throughout development, expressionwas cell type specific and temporally regulated (Table 2 see below).Elevated Magmas levels were found in cells and structures such asmyocytes, small bowel epithelium, kidney proximal tubules, nasal mucosaand salivary gland during embryogenesis.

In adult tissues, Magmas expression was also high in the epithelialcells as well as muscle. Epithelial cells are involved in protein andelectrolyte transport and have high metabolic requirements. Magmasexpression in these cells appears to correlate with function.

This relationship is illustrated by the staining pattern of thedeveloping renal structures and the choroid plexus. In the day 14.5embryo, there is no staining of the mesonephros, which containsprimative glomeruli and collecting tubules. Weak Magmas expressionbecomes apparent (day 15.5) with the formation of increasing numbers ofproximal and distal tubules and the initiation of urine production.Intense staining of the kidney is seen on day 18, when the organ isfully functional. In the choroid plexus, the detection of Magmas at day14.5 correlates with the production of cerebral spinal fluid by thisstructure [Catala, 1998 #113][Dziegielewska, 2001 #112]. Many of theepithelial cells mentioned are affected by diabetes.

Immunohistochemistry was done on sections of brain from patients withAlzheimer's Disease (AD) and we found surprisingly, that there isreduced Magmas in the pyramidal neurons of the hippocampus. These arethe cells known to be abnormal in the AD patients.

Besides ANT3, we have identified several other mitochondrial proteinsthat associate with Magmas.

In addition, upon screening for mutations in the different patientmaterials, we identified a Magmas point mutation in a patient with amitochondrial myopathy. This patient also has an unusual mitochondrialdefect because the activity of a mitochondrial enzyme, ATP synthetase,was elevated rather than reduced. ATP synthetase is one of the proteinsfound to associate with Magmas.

In addition, Magmas appears to interact with 3 ribosomal proteins,particularly ribosomal subunit S19, suggesting a potential role intranslation.

We have also identified mutations in the blasts of the diagnostic bonemarrow aspirations from patients with leukemia. All analysis done onsamples obtained before therapeutic interventions on primary diagnosticbone marrow sample. In 38 AML samples, 3 deletions and 4 insertions wereidentified. In 68 ALL samples, one deletion and two insertions in Magmaswere identified. In 66 other tumor samples and 52 normal samples, noabnormalities could be identified.

Mutations Identified AML Samples:

MAKYLAQIUVMGVQVVGRAFARALRQEFAASRFQPLRPQPPGGTAD SQRVQAEPZ (SEQ ID NO: 6),deletion of 14 codons results in 54 amino acid protein; andMAKYLAQIIVMGVQVVGRAFARALRQEFAASRRHSRFSTCPSZ (SEQ ID NO: 7), frameshiftdeletion of 22 codons results in 44 amino acid protein.

Mutation Identified in Four AML and Two ALL Samples:

MAKYLAQIVMGVQVVGRAFARALRQEFAASTPZ (SEQ ID NO: 8), a 174 bp insertresults in truncated 32 amino acid protein instead of 125 aa protein;and

Mutation Identified in an ALL Sample:

MAKYLAQIIVMGVQVVGRAFARALRQEFAASRAEADARGRAGHRSAAASNLSGLSLQEAQQKNYEHLFKVNDKSVGGSFYLQTKVVRAKERLDEELKIQAQ EDRKKGQMPHTZ (SEQ IDNO 9), a deletion of 13 amino acids, numbers 62-74 results in 112 aminoacid protein.

Magmas sequence analysis of individuals with mitochondrial defectsrevealed a patient with a glutamate->glycine mutation at amino acid 72od SEQ ID NO: 4. The mutation was present in one allele only. Thispatient has increased Complex V activity. Clinically the patient has anencephalomyopathy.

TABLE 2 Magmas expression during murine embryogenesis. Tissue 6.5 d 10.0d 12.5 d 14.5 d 15.5 d 18 d Trophoblast + + + ND ND ND Decidualcells + + + ND ND ND Yolk sac + + ND ND ND Embryonic + + + + weak weakectoderm Embryonic + Endoderm Somites ND + Cardiac muscle weak + weak ND− Skeletal muscle + + + − Spinal cord weak + weak ND ND Spinal gangliaweak + + ND weak Neural tube/Brain weak + weak weak weak Choroid plexusND weak weak weak Intestine weak + + weak weak Bronchials + + weak −Liver weak + + + − Salivary gland ND + Nasal mucosa − weak + Kidney ND −weak + Adrenal medulla ND +

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All references described herein and throughout the specification areincorporated herein by reference in their entirety.

1-10. (canceled)
 11. A method of detecting cancer comprising the stepsof: a) providing a biological sample from an individual suspected ofhaving cancer; b) analyzing the Magmas protein expression in the sample;and c) comparing the expression of Magmas protein in the sample againsta normal control, wherein increased expression of the Magmas protein inthe sample indicates that the individual is at increased risk forcancer.
 12. The method of claim 11, wherein the cancer is prostatecancer, neuroblastoma, Ewings sarcoma, osteosarcoma or leukemia.
 13. Themethod of claim 12, wherein the leukemia is acute myeloid leukemia (AML)or acute lymphoid leukemia (ALL).
 14. The method of claim 11, whereinthe cancer is prostate cancer.
 15. The method of claim 11, whereinanalyzing the Magmas protein expression is performed using ananti-Magmas antibody.
 16. The method of claim 15, wherein the measuringis performed in vivo by injecting a Magmas protein recognizing antibodyconnected to a detectable label into the individual.
 17. A method oftreating an individual affected with cancer comprising the steps ofadministering a therapeutic Magmas blocking amount of a compound thatinteracts with Magmas such as a Magmas antibody in a pharmaceuticallyacceptable carrier to an individual having cancer.
 18. The method ofclaim 17, wherein the cancer is prostate cancer, neuroblastoma, Ewingssarcoma, osteosarcoma or leukemia.
 19. The method of claim 17 where thecompound is antisense RNA, ribozymes, RNA decoys, antibodies, ordominant-negative mutants.
 20. A Diagnostic kit for Western blotting orimmunohistochemistry to detect the presence of cells expressing Magmasand to quantity the level of expression, containing immunoglobulinspecific for human MAGMAS.